Methods and reagents for discovering and using mammalian melanocortin receptor agonists and antagonists to modulate feeding behavior in animals

ABSTRACT

The present invention provides recombinant expression constructs comprising nucleic acid encoding mammalian melanocortin receptors, and mammalian cells into which said recombinant expression constructs have been introduced that express functional mammalian melanocortin receptors. The invention provides a panel of such transformed mammalian cells expressing melanocortin receptors for screening compounds for receptor agonist and antagonist activity. The invention also provides methods for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which are associated with the capacity to influence or modify metabolism and behavior, particularly feeding behavior.

This invention was made with government support under R29DK41921,R01DK43859, P01DK44239, AR42415 and HD30236 by the National Institutesof Health. The government has certain rights in the invention.

This application is a continuation-in-part of U.S. Ser. No. 08/466,906,filed Jun. 6, 1995, now U.S. Pat. No. 5,849,871 which is a divisional ofU.S. Ser. No. 07/886,979, filed May. 21, 1992, now U.S. Pat. No.5,270,605, issued Dec. 14 1993. This application is also acontinuation-in-part of U.S. Ser. No. 08/478,992, filed Jun. 7, 1995,now U.S. Pat. No. 5,773,229, which is a divisional of U.S. Ser. No.08/077,673, filed Jun. 15, 1993, now U.S. Pat. No. 5,554,729, which is adivisional of U.S. Ser. No. 07/866,560, filed Apr. 10, 1992, now U.S.Pat. No. 5,280,112, issued Jan. 18, 1994. This application is also acontinuation-in-part of U.S. Ser. No. 08/044,812, filed Apr. 8, 1993,now U.S. Pat. No. 5,837,521.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the cloning, expression and functionalcharacterization of mammalian melanocortin receptor genes. The inventionprovides nucleic acid encoding mammalian melanocortin receptors,recombinant expression constructs comprising said nucleic acid, andmammalian cells into which said recombinant expression constructs havebeen introduced, and that express functional mammalian melanocortinreceptors. The invention also provides a panel of such transformedmammalian cells expressing melanocortin receptors for screeningcompounds for receptor agonist and antagonist activity. The inventionprovides methods for using such panels of melanocortinreceptor-expressing mammalian cells to specifically detect and identifyagonists and antagonists for each melanocortin receptor, as well aspatterns of agonist and antagonist activity of said compounds for theclass of melanocortin receptors. Such screening methods provide a meansfor identifying compounds with patterns of melanocortin agonist andantagonist activity which are associated with the capacity to influenceor modify physiological funtion and behavior, particularly metabolismand feeding behavior.

2. Background of the Invention

The proopiomelanocortin (POMC) gene product is processed to produce alarge number of biologically active peptides. Two of these peptides,α-melanocyte stimulating hormone (αMSH), and adrenocorticotropic hormone(ACTH) have well understood roles in control of melanocyte andadrenocortical function, respectively. Both of these hormones are alsofound in a variety of forms with unknown functions, for example,γ-melanocyte stimulating hormone (γMSH), which has little or no abilityto stimulate pigmentation (Ling et al., 1979, Life Sci. 25: 1773-1780;Slominski et al., 1992, Life Sci. 50: 1103-1108). A melanocortinreceptor gene specific for each of the αMSH, ACTH and γMSH hormones hasbeen discovered by some of the present inventors (see U.S. Pat. Nos.5,280,112, 5,532,347 and U.S. application Ser. No. 08/044,812,incorporated by reference herein). In addition, two other melanocortinreceptor genes have been discovered by some of the present inventors(see Lu et al, 1994, Nature 371: 799-802; Mountjoy et al, 1994, Molec.Endocrinol. 8: 1298-1308) and others (see Gantz et al., 1993, J. Biol.Chem. 268: 15174-15179 and Labbe et al., 1994, Biochem. 33: 4543-4549).

Along with the well-recognized activities of αMSH in melanocytes andACTH in adrenal and pituitary glands, the melanocortin peptides alsohave a diverse array of biological activities in other tissues,including the brain and immune system, and bind to specific receptors inthese tissues with a distinct pharmacology (see, Hanneman et al., inPeptide Hormone as Prohormones, G. Martinez, ed. (Ellis Horwood Ltd.:Chichester, UK) pp. 53-82; DeWied & Jolles, 1982, Physiol. Rev. 62:976-1059 for reviews). A complete understanding of these peptides andtheir diverse biological activities requires the isolation andcharacterization of their corresponding receptors. Some biochemicalstudies have been reported in the prior art.

Shimuze, 1985, Yale J. Biol. Med. 58: 561-570 discusses the physiologyof melanocyte stimulating hormone.

Tatro & Reichlin, 1987, Endocrinology 121: 1900-1907 disclose that MSHreceptors are widely distributed in rodent tissues.

Sola et al., 1989, J. Biol. Chem. 264: 14277-14280 disclose themolecular weight characterization of mouse and human MSH receptorslinked to radioactively and photoaffinity labeled MSH analogues.

Siegrist et al., 1991, J. Receptor Res. 11: 323-331 disclose thequantification of receptors on mouse melanoma tissue by receptorautoradiography.

Cone & Mountjoy, U.S. patent application Ser. No. 07/866,979, filed Apr.10, 1992, disclose the isolation of human and mouse α-MSH receptor genesand uses thereof (incorporated herein by reference).

Cone & Mountjoy, U.S. patent application Ser. No. 07/866,560, filed Apr.10, 1992, disclose the isolation of human and bovine ACTH receptor genesand uses thereof (incorporated herein by reference).

Mountjoy et al., 1992, Science 257: 1248-1251 disclose the isolation ofcDNAs encoding mammalian ACTH and MSH receptor proteins.

POMC neurons are present in only two regions of the brain, the arcuatenucleus of the hypothalamus, and the nucleus of the solitary tract ofthe brain stem. Neurons from both sites project to a number ofhypothalamic nuclei known to be important in feeding behavior, includingthe paraventricular nucleus, lateral hypothalamic area, and ventromedialhypothalamic nucleus. While previous reports have claimed bothstimulatory and inhibitory effects of α-MSH on feeding behavior (seeShimizu et al., 1989, Life Sci. 45: 543-552; Tsujii et al., 1989, BrianRes. Bull. 23: 165-169), knowledge of specific melanocortin receptors,their location within the central nervous system and the necessarypharmacological tools were not sufficiently developed at that time toallow the resolution of this issue. The present inventors have shownherein that a novel antagonist of the MC-3 and MC-4 melanocortinreceptors can substantially increase food consumption in animals engagedin normal or fast-induced feeding behavior. This is consistent withexpression of both MC-3 and MC-4 receptor mRNAs at these sites in insitu hybridization studies (Roselli-Rehfuss et al., 1993, Proc. Natl.Acad. Sci. USA 90: 8856-8860; Mountjoy et al., 1994, Molec. Endocrinol.8: 1298-1308). Moreover, the regulation of arcuate nucleus POMC geneexpression is consistent with an inhibitory role for POMC in feedingbehavior. POMC mRNA levels are decreased following a fast (Bergendahl etal., 1992, Neuroendocrinol. 56: 913-920; Brady et al., 1990,Neuroendocrinol. 52: 441-447), and a significant diurnal variation inPOMC mRNA levels in the arcuate nucleus is seen in rat, with the nadiroccurring around the onset of nighttime feeding at 1800 hrs (Steiner etal., 1994, FASEB J. 8: 479-488).

Thus, the experimental evidence indicates that POMC neurons play animportant role in tonic inhibition of feeding behavior, wherein obesityresults from a chronic disruption of this inhibitory tone by antagonismof central melanocortin receptors in at least one animal model (agouti).

These results reveal for the first time a need in the art for a meansfor characterizing mammalian melanocortin receptor agonists andantagonists in vitro for the development of compounds that affectfeeding behavior in animals.

SUMMARY OF THE INVENTION

The present invention provides a biological screening system foridentifying and characterizing compounds that are agonists orantagonists of mammalian melanocortin receptors. The biologicalscreening system of the invention comprises a panel of transformedmammalian cells comprising a recombinant expression construct encoding amammalian melanocortin receptor, and expressing said receptor thereby.The invention provides such a panel of transformed mammalian cellswherein the panel comprises cells expressing each type of mammalianmelanocortin receptor. Thus, the invention also provides nucleic acidencoding mammalian melanocortin receptors, recombinant expressionconstructs comprising said nucleic acid, and mammalian cells into whichsaid recombinant expression constructs have been introduced, and thatexpress functional mammalian melanocortin receptors. Methods for usingsuch panels of melanocortin receptor-expressing mammalian cells tospecifically detect and identify agonists and antagonists for eachmelanocortin receptor, as well as patterns of agonist and antagonistactivity of said compounds for the class of melanocortin receptors, arealso provided. Such screening methods provide a means for identifyingcompounds with patterns of melanocortin agonist and antagonist activitywhich are associated with the capacity to influence or modify metabolismand behavior in an animal, particularly feeding behavior.

Thus, the invention provides in a first aspect a biological screeningpanel for determining the melanocortin receptor agonist/antagonistprofile of a test compound. The panel comprises a first mammalian cellcomprising a recombinant expression construct encoding a mammalianmelanocortin receptor that is the α-MSH (MC-1) receptor. The panel alsocomprises a second mammalian cell comprising a recombinant expressionconstruct encoding a mammalian melanocortin receptor that is the ACTH(MC-2) receptor. The panel also comprises a third mammalian cellcomprising a recombinant expression construct encoding a mammalianmelanocortin receptor that is the MC-3 receptor. The panel alsocomprises a fourth mammalian cell comprising a recombinant expressionconstruct encoding a mammalian melanocortin receptor that is the MC-4receptor. The panel also comprises a fifth mammalian cell comprising arecombinant expression construct encoding a mammalian melanocortinreceptor that is the MC-5 receptor. As provided by the invention, eachmammalian cell expresses the melanocortin receptor encoded by therecombinant expression construct comprised by said cell.

In preferred embodiments, the melanocortin receptors encoded by therecombinant expression constructs comprising the transformed mammaliancells comprising the panel are mouse MC-1 receptor (SEQ ID Nos.: 3 and4); human MC-1 receptor (SEQ ID Nos.: 5 and 6), human MC-2 (ACTH)receptor (SEQ ID Nos.: 7 and 8), bovine MC-2 receptor (SEQ ID Nos.: 9and 10), rat MC-3 receptor (SEQ ID Nos.: 11 and 12), human MC-4 receptor(SEQ ID Nos.: 15 and 16) and human MC-5 receptor (SEQ ID Nos.: 17 and18).

In a second aspect, the invention provides a method for using themelanocortin receptor panel to identify and characterize test compoundsas melanocortin receptor agonists and/or antagonists. In thisembodiment, the method provided by the invention identifies amelanocortin receptor agonist, and comprises the steps of contactingeach of the cells of the panel with a test compound to be characterizedas an agonist of a mammalian melanocortin receptor and detecting bindingof the test compound to each of the mammalian melanocortin receptors byassaying for a metabolite produced in the cells that bind the compound.In a preferred embodiment, the detected metabolite is cAMP.

In a preferred embodiment of this method, each of the cells of the panelof mammalian cells expressing mammalian melanocortin receptors furthercomprises a recombinant expression construct encoding a cyclic AMPresponsive element (CRE) transcription factor binding site that isoperatively linked to a nucleic acid sequence encoding a protein capableof producing a detectable metabolite. In preferred embodiments, saidprotein is β-galactosidase, most preferably encoded by a nucleic acidcomprising the recombinant expression construct identified aspCRE/β-galactosidase (as disclosed in Chen et al., 1994, Analyt.Biochem. 226: 349-354). As provided by the invention, expression of theprotein that produces the detectable metabolite is dependent on bindingof the test compound to the melanocortin receptor expressed by each cellin the panel and the intracellular production of cAMP as a result. Inthis embodiment, cAMP production results in expression of a proteincapable of producing a detectable metabolite, the protein mostpreferably being β-galactosidase. In preferred embodiments, thedetectable metabolite absorbs light to produce a colored product. Thus,this embodiment of the invention provides a panel of melanocortinreceptor-expressing cells whereby melanocortin hormone binding resultsin the production of a colored product in proportion to the extent ofcAMP production in the cell as a result of hormone receptor binding.

In another embodiment of this aspect of the invention is provided amethod for characterizing a compound as an antagonist of a mammalianmelanocortin receptor. In this embodiment, the method comprises thesteps of contacting each of the cells of the panel with an agonist ofthe mammalian melanocortin receptor in an amount sufficient to produce adetectable amount of a metabolite produced in the cells that bind theagonist, in the presence or absence of a test compound to becharacterized as an antagonist of a mammalian melanocortin receptor, anddetecting the amount of the metabolite produced in each cell in thepanel in the presence of the test compound with the amount of themetabolite produced in each cell in the panel in the absence of the testcompound. As provided by the assay, inhibition of the production of thedetectable metabolite is used as an indication that the tested compoundis a melanocortin receptor antagonist, which is further characterizedquantitatively by the extent of said inhibition.

In a preferred embodiment of this method, each of the cells of the panelof mammalian cells expressing mammalian melanocortin receptors furthercomprises a recombinant expression construct encoding a cyclic AMPresponsive element (CRE) transcription factor binding site that isoperatively linked to a nucleic acid sequence encoding a protein capableof producing a detectable metabolite. In preferred embodiments, saidprotein is β-galactosidase, most preferably encoded by a nucleic acidcomprising the recombinant expression construct identified aspCRE/β-galactosidase. As provided by the invention, expression of theprotein that produces the detectable metabolite is dependent on bindingof the test compound to the melanocortin receptor expressed by each cellin the panel. In preferred embodiments, the detectable metaboliteabsorbs light to produce a colored product. Thus, this embodiment of theinvention provides a panel of melanocortin receptor-expressing cellswhereby melanocortin hormone binding results in the production of acolored product in proportion to the extent of cAMP production in thecell as a result of hormone receptor binding.

The invention also provides melanocortin receptor agonists identified bythe methods and using the screening panel of the invention. In preferredembodiments, the agonist is an agonist of the MC-3 mammalianmelanocortin receptor. In preferred embodiments, the agonist is anagonist of the MC-4 mammalian melanocortin receptor.

The invention provides melanocortin receptor antagonists identified bythe methods and using the screening panel of the invention. In preferredembodiments, the antagonist is an antagonist of the MC-3 mammalianmelanocortin receptor. In preferred embodiments, the antagonist is anantagonist of the MC-4 mammalian melanocortin receptor.

The invention also provides methods for characterizing mammalianmelanocortin receptor agonists for the capacity to modify or influencemetabolism and feeding behavior in an animal. In a first aspect, theinvention provides a method for characterizing melanocortin receptorMC-3 or MC-4 agonists as inhibitors of feeding behavior in an animal,the method comprising the steps of providing food to an animal that hasbeen deprived of food for at least 12 hours, with or withoutadministering to the animal an MC-3 or MC-4 receptor agonist of theinvention, and comparing the amount of food eaten by the animal afteradministration of the MC-3 or MC-4 receptor agonist with the amount offood eaten by the animal without administration of the MC-3 or MC-4receptor agonist.

In another aspect, the invention provides a method for characterizing amelanocortin MC-3 or MC-4 receptor antagonist as a stimulator of feedingbehavior in an animal. In this embodiment, the method comprises thesteps of providing food to an animal not deprived of food for at least12 hours, with or without administering to the animal an MC-3 or MC-4receptor antagonist, immediately prior to the onset of darkness ornighttime, and comparing the amount of food eaten by the animal afteradministration of the MC-3 or MC-4 receptor antagonist with the amountof food eaten by the animal without administration of the MC-3 or MC-4receptor antagonist.

Thus, the invention also provides methods for using certain of themelanocortin receptor agonists and antagonists for modifying feedingbehavior in an animal. In a first aspect, the invention provides amethod for stimulating feeding in an animal, the method comprisingadministering to the animal an MC-3 or MC-4 receptor antagonist. In apreferred embodiment, the antagonists are administered systemically. Inadditional embodiments, the antagonists are administeredintracerebroventricularly.

In another aspect, the invention provides a method for inhibitingfeeding in an animal, the method comprising administering to the animalan MC-3 or MC-4 receptor agonist. In a preferred embodiment, theagonists are administered systemically. In additional embodiments, theagonists are administered intracerebroventricularly.

In yet another aspect, the invention provides mammalian melanocortinreceptor agonists having the general formula:

    A-B-C-D-E-F-G-amide

wherein A is an aliphatic amino acid residue, including for example Leu,Ile, Nle and Met, as well as analogues and substituted derivativesthereof; B is an acidic amino acid residue, including for example Aspand Glu; C is a basic amino acid residue, such as His; D is an aromaticamino acid residue having a D- conformation, including D-Phe, D-Tyr andsubstituted derivatives thereof; E is a basic amino acid residue, forexample Arg, Lys, homoArg, homoLys, and analogues or substitutedderivatives thereof; F is Trp or substituted derivatives thereof; and Gis Lys, homoLys or a substituted derivative thereof. In the peptideembodiments of the melanocortin receptor agonists of the invention, thepeptide is cyclized by the formation of an amide bond between the sidechain carboxyl group of the Asp or Glu residue at position B in thepeptide, and the side chain amino group of the Lys or homoLys residue atposition G. In preferred embodiments, the melanocortin receptor agonistsof the invention are agonists of the MC-3 or MC-4 receptor.

The invention also provides mammalian melanocortin receptor antagonistshaving the general formula:

    A-B-C-D-E-F-G-amide

wherein A is an aliphatic amino acid residue, including for example Leu,Ile, Nle and Met, as well as analogues and substituted derivativesthereof; B is an acidic amino acid residue, including for example Aspand Glu; C is a basic amino acid residue, such as His; D is an aromaticamino acid residue having a D- conformation, including D-Nal andsubstituted derivatives thereof; E is a basic amino acid residue, forexample Arg, Lys, homoArg, homoLys, and analogues or substitutedderivatives thereof; F is Trp or substituted derivatives thereof; and Gis Lys, homoLys or a substituted derivative thereof. In the peptideembodiments of the melanocortin receptor antagonists of the invention,the peptide is cyclized by the formation of an amide bond between theside chain carboxyl group of the Asp or Glu residue at position B in thepeptide, and the side chain amino group of the Lys or homoLys residue atposition G. In preferred embodiments, the melanocortin receptorantagonists of the invention are agonists of the MC-3 or MC-4 receptor.

It is an advantage of the present invention that it provides an in vitroscreening method for characterizing compounds having melanocortinreceptor binding activities that relate to feeding behavior in animals.Specifically, the invention advantageously provides means and methodsfor identifying compounds having melanocortin receptor agonist and/orantagonist activity that have been associated with either stimulating orinhibiting feeding behavior when administered to an animal. Theinvention thus provides an economical first step in screening compoundsfor the capacity to affect feeding behavior, including synthetic,peptidomimetic or organomimetic derivatives of melanocortin receptoragonists or antagonists as disclosed herein or elsewhere.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the nucleotide (SEQ ID No.: 3) and amino acid(SEQ ID No.: 4) sequence of the mouse melanocyte stimulating hormonereceptor gene.

FIGS. 2A and 2B illustrate the nucleotide (SEQ ID No.: 5) and amino acid(SEQ ID No.: 6) sequence of the human melanocyte stimulating hormonereceptor gene.

FIGS. 3A and 3B illustrate the nucleotide (SEQ ID No.: 7) and amino acid(SEQ ID No.: 8) sequence of the human adrenocorticotropic hormonereceptor gene.

FIGS. 4A and 4B illustrate the nucleotide (SEQ ID No.: 9) and amino acid(SEQ ID No.: 10) sequence of the bovine adrenocorticotropic hormonereceptor gene.

FIGS. 5A and 5B illustrate the nucleotide (SEQ ID No:11) and amino acid(SEQ ID No.:12) sequences of the rat melanocortin-3 receptor.

FIGS. 6A and 6B illustrate the nucleotide (SEQ ID No.: 15) and aminoacid (SEQ ID No.: 16) sequence of the human melanocortin 4 receptorgene.

FIGS. 7A and 7B illustrate the nucleotide (SEQ ID No: 17) and amino acid(SEQ ID No.: 18) sequences of the rat melanocortin-5 receptor gene.

FIG. 8 shows a graph of intracellular cAMP accumulation resulting frommelanocyte stimulating hormone receptor agonist binding in human 293cells transfected with a MSH receptor-encoding recombinant expressionconstruct.

FIG. 9 illustrates the cAMP response of mouse Y1 cells to binding ofmelanocortin peptides to human melanocortin-2 (ACTH) receptor, asmeasured using the β-galactosidase assay described in Example 4.

FIG. 10 illustrates the results of competition binding experiments ofmelanocortin peptides to cells expressing a recombinant expressionconstruct encoding the rat melanocortin-3 receptor.

FIGS. 11A through 11C illustrate the results of experiments showingintracellular cAMP accumulation caused by receptor-ligand binding inhuman 293 cells expressing the MC-3 receptor.

FIG. 12 shows a graph of intracellular cAMP accumulation resulting frompeptides to human melanocortin-4 receptor agonist binding in human 293cells transfected with a MC-4 receptor-encoding recombinant expressionconstruct.

FIG. 13 illustrates the results of cAMP accumulation (AC) andcAMP-dependent β-galactosidase (β-gal) assays of melanocortin peptidebinding to a rat melanocortin-5 receptor.

FIG. 14 illustrates the structure of the pCRE/β-gal plasmid.

FIGS. 15A through 15D illustrates the results of theβ-galactosidase-coupled, colorimetric melanocortin receptor bindingassay using cells expressing each of the MC-1, MC-3, MC-4 or MC-5receptors and contacted with αMSH or a variety of αMSH analogues.

FIGS. 16A through 16D shows the results of the β-galactosidase-coupled,colorimetric melanocortin receptor binding assay to determine antagonistactivity of melanocortin analogues SHU9119 and SHU8914 in cellsexpressing each of the melanocortin receptors MC-1, MC-3, MC-4 and MC-5.

FIGS. 17A and 17B shows the results of classic competition bindingassays using the melanocortin analogues SHU9119 and SHU8914 at the MC3Rand MC-4 R receptors.

FIGS. 18A and 18B shows the results of cAMP accumulation experiments(performed using the β-galactosidase assay of Example 4) for mouse MC-4receptor (FIG. 18A) and rat MC-5 receptor (FIG. 18B).

FIGS. 19A through 19C show the effect on food intake ofintracerebroventricular administration of melanocortin analogue SHU9119in mice.

FIGS. 20A through 20C show the effect on food intake ofintracerebroventricular administration of melanocortin analogue MTII inmice.

FIG. 20D shows the effect on locomotor activity ofintracerebroventricular administration of melanocortin analogue MTII inmice.

FIGS. 21A through 21D show the effect on food intake ofintracerebroventricular administration of melanocortin analogue MTII inmice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "melanocortin receptor" as used herein refers to proteinshaving the biological activity of any of the disclosed melanocortinreceptors, including the MC-1 (SEQ ID Nos.: 3, 4, 5 and 6), MC-2 (ACTH;SEQ ID Nos.: 7, 8, 9 and 10), MC-3 (SEQ ID Nos.: 11 and 12), MC-4 (SEQID Nos.: 15 and 16) or MC-5 (SEQ ID Nos.: 17 and 18) receptors, as wellas naturally-occurring and genetically-engineered allelic variations inthese sequences.

Cloned nucleic acid provided by the present invention may encode MCreceptor protein of any species of origin, including, for example,mouse, rat, rabbit, cat, and human, but preferably the nucleic acidprovided by the invention encodes MC receptors of mammalian, mostpreferably rodent and human, origin.

The production of proteins such as the MC receptors from cloned genes bygenetic engineering means is well known in this art. The discussionwhich follows is accordingly intended as an overview of this field, andis not intended to reflect the full state of the art.

DNA which encodes MC receptors may be obtained, in view of the instantdisclosure, by chemical synthesis, by screening reverse transcripts ofmRNA from appropriate cells or cell line cultures, by screening genomiclibraries from appropriate cells, or by combinations of theseprocedures, as illustrated below. Screening of mRNA or genomic DNA maybe carried out with oligonucleotide probes generated from the MCreceptor gene sequence information provided herein. Probes may belabeled with a detectable group such as a fluorescent group, aradioactive atom or a chemiluminescent group in accordance with knowprocedures and used in conventional hybridization assays, as describedin greater detail in the Examples below. In the alternative, MC receptorgene sequences may be obtained by use of the polymerase chain reaction(PCR) procedure, with the PCR oligonucleotide primers being producedfrom the MC receptor gene sequences provided herein. See U.S. Pat. Nos.4,683,195 to Mullis et al. and 4,683,202 to Mullis.

MC receptor proteins may be synthesized in host cells transformed with arecombinant expression construct comprising a nucleic acid encoding eachof the receptors disclosed herein. Such a recombinant expressionconstruct can also be comprised of a vector that is a replicable DNAconstruct. Vectors are used herein either to amplify DNA encoding an MCreceptor and/or to express DNA which encodes an MC receptor. For thepurposes of this invention, a recombinant expression construct is areplicable DNA construct in which a DNA sequence encoding an MC receptoris operably linked to suitable control sequences capable of effectingthe expression of the receptor in a suitable host cell. The need forsuch control sequences will vary depending upon the host selected andthe transformation method chosen. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences which control the termination of transcription andtranslation. Amplification vectors do not require expression controldomains. All that is needed is the ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants. See, Sambrook et al., 1990,Molecular Cloning: A Laboratorv Manual (Cold Spring Harbor Press: NewYork).

Also specifically provided by the invention are reporter expressionconstructs comprising a nucleic acid encoding a protein capable ofexpressing a detectable phenotype, such as the production of adetectable reporter molecule, in a cell expressing the construct. Suchconstructs can be used for producing recombinant mammalian cell lines inwhich the reporter construct is stably expressed. Most preferably,however, the reporter construct is provided and used to induce transientexpression over an experimental period of from about 18 to 96 hrs inwhich detection of the reporter protein-produced detectable metabolitecomprises an assay. Such reporter expression constructs are alsoprovided wherein induction of expression of the reporter construct iscontrolled by a responsive element operatively linked to the codingsequence of the reporter protein, so that expression is induced onlyupon proper stimulation of the responsive element. Exemplary of such aresponsive element is a cAMP responsive element (CRE), which inducesexpression of the reporter protein as a result of an increase inintracellular cAMP concentration. In the context of the presentinvention, such a stimulus is associated with melanocortin receptorbinding, so that a reporter construct comprising one or more CREs isinduced to express the reporter protein upon binding of a receptoragonist to a MC receptor in a recombinantly transformed mammalian cell.Production and use of such a reporter construct is illustrated below inExample 5.

Vectors useful for practicing the present invention include plasmids,viruses (including phage), retroviruses, and integratable DNA fragments(i.e., fragments integratable into the host genome by homologousrecombination). The vector replicates and functions independently of thehost genome, or may, in some instances, integrate into the genomeitself. Suitable vectors will contain replicon and control sequenceswhich are derived from species compatible with the intended expressionhost. A preferred vector is the plasmid pcDNA/neo I. Transformed hostcells are cells which have been transformed or transfected withrecombinant expression constructs made using recombinant DNA techniquesand comprising mammalian MC receptor-encoding sequences. Preferred hostcells are human 293 cells. Preferred host cells for the MC-2 (ACTH)receptor are Y1 cells (subclone OS3 or Y6). Transformed host cells arechosen that ordinarily express functional MC receptor protein introducedusing the recombinant expression construct. When expressed, themammalian MC receptor protein will typically be located in the host cellmembrane. See, Sambrook et al., ibid.

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant MC receptor protein synthesis. In principal, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. However, mammalian cells are preferred, asillustrated in the Examples. Propagation of such cells in cell culturehas become a routine procedure. See Tissue Culture, Academic Press,Kruse & Patterson, editors (1973). Examples of useful host cell linesare human 293 cells, VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, mouse Y1 (subclone OS3), and WI138, BHK, COS-7, CV, and MDCKcell lines. Human 293 cells are preferred.

Cells expressing mammalian MC receptor proteins made from cloned genesin accordance with the present invention may be used for screeningagonist and antagonist compounds for MC receptor activity. Competitivebinding assays are well known in the art and are described in theExamples below. Such assays are useful for drug screening of MC receptoragonist and antagonist compounds, as detected in receptor binding assaysas described below.

One particular use of such screening assays is for developing drugs andother compounds useful in modifying or changing feeding behavior inmammals. The invention provides an assay system, comprising a panel ofrecombinant mammalian cells, heterologously expressing each of the MCreceptors disclosed herein, wherein the panel is constructed of at leastone cell line expressing an MC receptor. The invention provides suchpanels also comprising a detection means for detecting receptor agonistor antagonist binding, such as the reporter expression constructsdescribed herein, using direct binding and competition binding assays asdescribed in the Examples below. In the use of this panel, each MCreceptor is assayed for agonist or antagonist patterns of binding a testcompound, and a characteristic pattern of binding for all MC receptorsis thereby determined for each test compound. This pattern is thencompared with known MC receptor agonists and antagonists to identify newcompounds having a pattern of receptor binding activity associated witha particular behavioral or physiological effect.

For example, provided herein is experimental evidence that MC-3 or MC-4receptor antagonists are capable of stimulating feeding in satedanimals, and that MC-3 or MC-4 agonists are capable of inhibitingfeeding in animals otherwise stimulated to eat. The invention providesan in vitro assay to characterize MC-3 and MC-4 agonists/antagonists asa preliminary and economical step towards developing feedingbehavior-modulating drugs for use in vivo.

These results on feeding behavior in vivo have been obtained withcertain MC receptor binding analogues, SHU9119 and MTII. These compoundshave the following chemical structure: ##STR1## (An additional analogue,SHU8914, has been tested in in vitro receptor binding assays; thiscompound is identical in structure to SHU9119, except that the aminoacid analogue at position 7 is para-iodo-D-phenylalanine.)

Generally, those skilled in the art will recognize that peptides asdescribed herein may be modified by a variety of chemical techniques toproduce compounds having essentially the same activity as the unmodifiedpeptide, and optionally having other desirable properties. For example,carboxylic acid groups of the peptide, whether carboxyl-terminal orsidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁ -C₁₆ester, or converted to an amide of formula NR₁ R₂ wherein R₁ and R₂ areeach independently H or C₁ -C₁₆ alkyl, or combined to form aheterocyclic ring, such as 5- or 6-membered. Amino groups of thepeptide, whether amino-terminal or sidechain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁ -C₁₆ alkyl or dialkyl amino or furtherconverted to an amide. Hydroxyl groups of the peptide sidechain may beconverted to C₁ -C₁₆ alkoxy or to a C₁ -C₁₆ ester using well-recognizedtechniques. Phenyl and phenolic rings of the peptide sidechain may besubstituted with one or more halogen atoms, such as fluorine, chlorine,bromine or iodine, or with C₁ -C₁₆ alkyl, C₁ -C₁₆ alkoxy, carboxylicacids and esters thereof, or amides of such carboxylic acids. Methylenegroups of the peptide sidechains can be extended to homologous C₂ -C₄alkylenes. Thiols can be protected with any one of a number ofwell-recognized protecting groups, such as acetamide groups. Thoseskilled in the art will also recognize methods for introducing cyclicstructures into the peptides of this invention to select and provideconformational constraints to the structure that result in enhancedbinding and/or stability. For example, a carboxyl-terminal oramino-terminal cysteine residue can be added to the peptide, so thatwhen oxidized the peptide will contain a disulfide bond, therebygenerating a cyclic peptide. Other peptide cyclizing methods include theformation of thioethers and carboxyl- and amino-terminal amides andesters.

Peptidomimetic and organomimetic embodiments are also hereby explicitlydeclared to be within the scope of the present invention, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid sidechains in the peptide,resulting in such peptido- and organomimetics of the peptides of thisinvention having substantial biological activity. It is implied that apharmacophore exists for the receptor agonist and antagonist propertiesof these and related MC receptor binding analogues. A pharmacophore isan idealized, three-dimensional definition of the structuralrequirements for biological activity. Peptido- and organomimetics can bedesigned to fit each pharmacophore with current computer modelingsoftware (computer aided drug design). MC receptor binding analoguesderived using such software and comprising peptido- and organomimeticsof SHU9119 and MTII and related analogues are within the scope of theclaimed invention.

The MC receptor binding analogues, in particular those analogues thatare MC-3 or MC-4 receptor agonists or antagonists are provided to beused in methods of influencing, modifying or changing feeding behaviorin mammals in vivo. Specific examples of uses for the MC receptorbinding analogues of the invention include but are not limited totreatment of eating disorders such as anorexia and obesity, and otherpathological weight and eating-related disorders. Other examples arefailure to thrive disorders and disease-related cachexia, such as occursin cancer patients. Also within the scope of the analogues of theinvention is use for enhancing appearance, athletic ability, or adjuvantto other therapies to treat disorders such as high blood pressure, highserum cholesterol, vascular and heart disease, stroke, kidney disease,diabetes and other metabolic disorders.

The Examples which follow are illustrative of specific embodiments ofthe invention, and various uses thereof. They set forth for explanatorypurposes only, and are not to be taken as limiting the invention.

EXAMPLE 1 Isolation of an αMSH Receptor Probe by Random PCRAmplification of Human Melanoma cDNA Using Degenerate OligonucleotidePrimers

In order to clone novel G-protein coupled receptors, cDNA prepared fromRNA from human melanoma cells was used as template for a polymerasechain reaction (PCR)-based random cloning experiment. PCR was performedusing a pair of degenerate oligonucleotide primers corresponding to theputative third and sixth transmembrane regions of G-protein coupledreceptors (Libert et al., 1989, Science 244: 569-72; Zhou et al., 1990,Nature 347: 76-80). The PCR products obtained in this experiment werecharacterized by nucleotide sequencing. Two novel sequences representingnovel G-protein-coupled receptors were identified.

PCR amplification was performed as follows. Total RNA was isolated froma human melanoma tumor sample by the guanidinium thiocyanate method(Chirgwin et al., 1979, Biochemistry 18: 5294-5299). Double-strandedcDNA was synthesized from total RNA with murine reverse transcriptase(BRL, Gaithersburg, Md.) by oligo-dT priming (Sambrook et al., ibid.).The melanoma cDNA mixture was then subjected to 45 cycles of PCRamplification using 500 picomoles of degenerate oligonucleotide primershaving the following sequence:

    Primer III (sense):                                                           GAGTCGACCTGTG(C/T)G(C/T)(C/G)AT(C/T)(A/G)CIIT(G/T)GAC(C/A)G(C/G)TAC                                                     (SEQ ID NO:1)                       and                                                                           Primer VI (antisense):                                                        CAGAATTCAG(T/A)AGGGCAICCAGCAGAI(G/C)(G/A)(T/C)GAA                                                                       (SEQ ID NO:2)                   

in 100 μl of a solution containing 50 mM Tris-HCl (pH 8.3), 2.5 mMMgCl₂, 0.01% gelatin, 200 μM each dNTP, and 2.5 units of Taq polymerase(Saiki et al., 1988, Science 239: 487-491). These primers werecommercially synthesized by Research Genetics Inc. (Huntsville, Ala.).Each PCR amplification cycle consisted of incubations at 94° C. for 1min (denaturation), 45° C. for 2 min (annealing), and 72° C. for 2 min(extension).

Amplified products of the PCR reaction were extracted withphenol/chloroform and precipitated with ethanol. After digestion withEcoRl and SalI, the PCR products were separated on a 1.2% agarose gel. Aslice of this gel, corresponding to PCR products of 300 basepairs (bp)in size, was cut out and purified using glass beads and sodium iodide,and the insert was then cloned into a pBKS cloning vector (Stratagene,LaJolla, Calif.).

A total of 172 of such pBKS clones containing inserts were sequencedusing Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) by thedideoxynucleotide chain termination method (Sanger et al., 1977, Proc.Natl. Acad. Sci. USA 74: 5463-5467). Two types of sequences homologousto other G-protein coupled receptors were identified.

EXAMPLE 2A Isolation of a Mouse αMSH (MC-1) Receptor cDNA

Probes isolated in Example 1 were used to screen a Cloudman melanomacDNA library in order to isolate a full-length cDNA corresponding to thecloned probe. One clone was isolated from a library of 5×10⁶ clonesscreened as described in co-owned U.S. Pat. No. 5,532,347, incorporatedby reference. This clone contained an insert of 2.6 kilobases (kb). Thenucleotide sequence of the complete coding region was determined (seeco-owned U.S. Pat. No. 5,532,347, incorporated by reference); a portionof this cDNA comprising the coding region was sequenced and is shown inFIGS. 1A and 1B (SEQ ID Nos: 3 & 4).

EXAMPLE 2B Isolation of a Human αMSH (MC-1) Receptor cDNA

In order to isolate a human counterpart of the murine melanocyte αMSHreceptor gene disclosed in Example 2A and co-owned U.S. Pat. No.5,532,347, a human genomic library was screened at high stringency (50%formamide, 42° C.) using the human PCR fragments isolated as describedin Example 1. A genomic clone was determined to encode an human MSHreceptor (SEQ ID NO:5.; FIGS. 2A and 2B). The human MSH receptor has apredicted amino acid sequence (SEQ ID NO:6) that is 75% identical andcolinear with the mouse αMSH receptor cDNA sequence. The predictedmolecular weight of the human MSH receptor is 34.7 kD.

EXAMPLE 2C Isolation of a Human ACTH (MC-2) Receptor cDNA

For cloning the ACTH receptor (MC-2), a human genomic library wasscreened at high stringency (50% formamide, 1M NaCl, 50 nM Tris-HCl, pH7.5, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100 μg/mlsalmon sperm DNA, 10 ×Denhardt's solution, 42° C.), using the human PCRfragments isolated as described in Example 1 herein and U.S. Pat. No.5,280,112, incorporated by reference. A genomic clone was isolated thatencodes a highly related G-coupled receptor protein (SEQ ID No:7 andFIGS. 3A and 3B). The predicted amino acid sequence (SEQ ID NO:8) ofthis clone is 39% identical and also colinear, excluding the thirdintracellular loop and carboxy-terminal tail, with the human MSHreceptor gene product. The predicted molecular weight of this putativeACTH receptor is 33.9 kilodaltons (kD). This clone was identified asencoding an MC-2 receptor based on its high degree of homology to themurine and human MSH receptors, and the pattern of expression indifferent tissue types, as described in Example 3 in U.S. Pat. No.5,280,112, incorporated by reference herein.

EXAMPLE 2D Isolation of a Bovine ACTH (MC-2) Receptor cDNA

A bovine genomic DNA clone encoding the bovine counterpart of the MC-2(ACTH) receptor was isolated from a bovine genomic library, essentiallyas described in Example 2C above, and its nucleotide sequence determined(as shown in FIGS. 4A and 4B; SEQ ID Nos:9 & 10).

EXAMPLE 2E Isolation of a Rat γ-MSH (MC-3) Receptor cDNA

The mouse αMSH receptor cDNA isolated as described in Example 2A andco-owned U.S. Pat. No. 5,532,347 was used to screen a rat hypothalamuscDNA library at low stringency (30% formamide, 5×SSC, 0.1% sodiumpyrophosphate, 0.2% sodium dodecyl sulfate, 100 μg/ml salmon sperm DNA,and 10% Denhardt's solution) at 42° C. for 18 h. A 1 kb cDNA clone wasisolated and sequenced as described in co-owned U.S. Pat. No. 5,532,347,and this clone used to re-screen the rat hypothalamus cDNA library athigh stringency (same conditions as above except that formamide waspresent at 45%). A cDNA clone approximately 2.0 kb in length wasisolated and analyzed as described in co-pending U.S. application Ser.No. 08/044,812, incorporated by reference; a portion of this cDNAcomprising the coding region was sequenced and is shown in FIGS. 5A and5B (SEQ ID Nos:11 & 12).

EXAMPLE 2F Isolation of a Human MC-4 Receptor DNA

For cloning the MC-4 receptor, a human genomic library was screened atmoderate stringency (40% formamide, 1M NaCl, 50 mM Tris-HCl, pH 7.5,0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulfate, 100 μg/ml salmonsperm DNA, 10 ×Denhardt's solution, 42° C.), using rat PCR fragmentsisolated as described in Example 1 herein, with the exception that thefollowing primers were used for PCR:

    Primer II (sense):                                                            GAGTCGACC(A/G)CCCATGTA(C/T)T(AGT)(C/T)TTCATCTG                                                             (SEQ ID NO:13)                                   and                                                                           Primer VII (antisense):                                                       CAGAATTCGGAA(A/G)GC(A/G)TA(G/T)ATGA(A/G)GGGGTC                                                             (SEQ ID NO:14)                               

A genomic clone was isolated that encodes a highly related G-coupledreceptor protein (SEQ ID NO:15 and FIGS. 6A and 6B) on a 1.9 kb HindIIIfragment. The predicted amino acid sequence (SEQ ID NO:16) of this cloneshares 55-61% sequence identity with human MC-3 and MC-5 receptors, and46-47% sequence identity with the human MC-1 and MC-2 (ACTH) receptor.

EXAMPLE 2G Isolation of a Mouse MC-5 Receptor DNA

One million clones from a mouse 129SVJ genomic library comprising5,000,000 clones in the λFixII vector (Stratagene) were screened at lowstringency (hybridization in 40% formamide at 42° C., washing performedin 0.5×SSC at 60° C., as described above in Example 2E) usingradiolabeled probes from the rat MC-3 and MC-4 receptors, as describedin Examples 2E and 2F. Positively-hybridizing clones were isolated andsequenced, and the sequences obtained were compared topreviously-isolated melanocortin receptor clones. One clone, comprisinga previously-unknown sequence, was determined to encode the MC-5melanocortin receptor. The nucleotide and amino acid sequences of thisreceptor are shown in FIGS. 7A and 7B (SEQ ID No.: 17 & 18).

EXAMPLE 3 Construction of a Recombinant Expression Construct, DNATransfection and Functional Expression of the MCR Gene Products

In order to produce recombinant mammalian cells expressing each of themelanocortin receptors of Example 2, cDNA or the coding exons fromgenomic DNA from each receptor was cloned into a mammalian expressionconstruct, the resulting recombinant expression construct transfectedinto human 293 cells, and cell lines generated that expressed themelanocortin receptor proteins in cellular membranes at the cellsurface.

The mouse αMSH receptor was cloned by excising the entire coding regionof the αMSH^(R) (MC-1) cDNA insert comprising a 2.1 kb fragment andsubcloning this fragment into the BamHI/XhoI sites of pcDNAI/neoexpression vector (Invitrogen, San Diego, Calif.). The resulting plasmidwas prepared in large-scale through one cycle of CsCl gradientultracentrifugation, and 20 μg of the plasmid transfected into each 100mm dish of 293 cells using the calcium phosphate method (see Chen &Okayama, 1987, Mol. Cell. Biol. 7: 2745-2752). After transfection, cellswere cultured in DMEM media supplemented with 10% calf serum in a 3% CO₂atmosphere at 37° C. Selection was performed with neomycin (G418; GIBCO)at a concentration of 1000 μg/mL; selection was started 72 hr aftertransfection and continued for 3 weeks.

The αMSH receptor is known to couple to G-proteins and thereby activateadenyl cyclase, increasing intracellular levels of cAMP (see Buckley &Ramachandran, 1981, Proc. Natl. Acad. Sci. USA 78: 7431-7435;Grahame-Smith et al., 1967, J. Biol. Chem 242: 5535-5541; Mertz & Catt,1991, Proc. Natl. Acad. Sci. USA 88: 8525-8529; Pawalek et al., 1976,Invest. Dermatol. 66: 200-209). This property of cells expressing theαMSH receptor was used analyze expression of the αMSH receptor in cellcolonies transfected with the expression vectors described herein asfollows. Cells (˜1×10⁶) were plated in 6-well dishes, washed once withDMEM containing 1% bovine serum albumin (BSA) and 0.5 mM IBMX (aphosphodiesterase inhibitor), then incubated for 45 minutes at 37° C.with varying concentrations of the melanotropic peptides αMSH, βMSH,γMSH, the MSH peptide analogues Nle⁴, D-Phe⁷ -αMSH (NDP-MSH), and ACTH.Following hormone treatment, the cells were washed twice with phosphatebuffered saline and intracellular cAMP extracted by lysing the cellswith 1 mL of 60% ethanol. Intracellular cAMP concentrations weredetermined using an assay (Amersham) which measures the ability of cAMPto displace (8-³ H) cAMP from a high affinity cAMP binding protein (seeGilman, 1970, Proc. Natl. Acad. Sci. USA 67: 305-312).

The results of these experiments are shown in FIG. 8. The abscissaindicates the concentration of each hormone and the ordinate indicatesthe percentage of basal intracellular cAMP concentration achieved byeach treatment. Points indicate the mean of duplicate incubations; thestandard error did not exceed 15% for any data point. None of thepeptides tested induced any change in intracellular cAMP in cellscontaining the vector alone. Cells expressing the murine αMSH receptorresponded to melanotropic peptides with a 2-3 fold elevation ofintracellular cAMP, similar to levels of cAMP induced by these peptidesin the Cloudman cell line (see Pawalek, 1985, Yale J. Biol. Med. 58:571-578). The EC₅₀ values determined for αMSH (2.0×10⁻⁹ M), ACTH(8.0×10⁻⁹ M) and the superpotent MSH analogue NDP-MSH (2.8×10⁻¹¹ M)correspond closely to reported values (see Tatro et al., 1990, CancerRes. 50: 1237-1242). As expected, the βMSH peptide had an EC₅₀ valuecomparable to αMSH, while γMSH had little or no activity (see Slominskiet al., 1992, Life Sci. 50: 1103-1108), confirming the identity of thisreceptor as a melanocyte αMSH receptor.

A similar series of experiments were performed using mouse Y1 cells(subclone OS3; Schimmer et al., 1995, J. Cell. Physiol. 163: 164-171)expressing the human and bovine MC-2 (ACTH) receptor clones of Examples2C and 2D. These results are shown in FIG. 9, where the extent of cAMPresponsive element-linked β-galactosidase activity (see Example 4,below) is shown with increasing concentrations of ACTH.

The entire coding region of the MC-3 receptor cDNA insert, obtained asdescribed above in the copending U.S. Ser. No. 08/044,812, was containedin a 2.0 kb restriction enzyme digestion fragment and was cloned intothe BamHI/XhoI sites of pcDNA/neo I expression vector (Invitrogen, SanDiego, Calif.). The resulting plasmid was prepared in large-scalethrough one cycle of CsCl gradient ultracentrifugation and 20 μgpcDNA/MC-3 receptor DNA were transfected into each 100 mm dish of 293cells using the calcium phosphate method (see Chen & Okayama, 1987, Mol.Cell. Biol. 7: 2745-2752). After transfection, cells were cultured inDMEM media supplemented with 10% calf serum in a 3% CO₂ atmosphere at37° C. Selection was performed with neomycin (G418; GIBCO) at aconcentration of 1000 μg/mL; selection was started 72 h aftertransfection and continued for 3 weeks.

Specific binding of melanocortin peptides to cells expressing the MC-3receptor was demonstrated by competition experiments using ¹²⁵ I-labeledNle⁴ -D-Phe⁷ -α-MSH (NDP-MSH, as described in Tatro et al., 1990, CancerRes. 50: 1237-1242). Suspended cells (2×10⁵) were incubated at 37° C.with 500,000 cpm of labeled peptide for 10 min in binding buffer (Ham'sF10 media plus 10 mM HEPES, pH 7.2, 0.25% bovine serum albumin, 500KIU/mL aprotinin, 100 μg/mL bacitracin and 1 mM 1,10-phenanthroline) inthe presence or absence of the indicated concentrations of peptides.Maximum labeling was achieved within 10 min.

The results of these experiments are shown in FIG. 10. Labeled NDP-MSHbinding to cells expressing the MC-3 receptor, produced as describedabove, is inhibited by competition with unlabeled peptides known to bemelanocortin receptor agonists, having a relative order of potency asfollows:

    NDP-MSH>γ-MSH>α-MSH>ACTH.sub.4-10 >>>ORG2766.

Approximate K_(i) values derived from this experiment are as shown inTable I:

                  TABLE I                                                         ______________________________________                                        Agonist       K.sub.i (approx)                                                ______________________________________                                        NDP-MSH       2 × 10.sup.-8                                             γ-MSH   5 × 10.sup.-8                                             α-MSH   1 × 10.sup.-7                                             ACTH.sub.4-10 8 × 10.sup.-5                                             ______________________________________                                    

cAMP production assays as described above were also used to analyzeexpression of MC-3R in cells transfected with the expression vectorsdescribed herein as follows. Cells (˜5×10⁶) were plated in 6-welldishes, washed once with DMEM containing 1% bovine serum albumin (BSA)and 0.5 mM IBMX (a phosphodiesterase inhibitor), then incubated for 1 hat 37° C. with varying concentrations of the melanotropic peptides αMSH,γ₃ MSH, γMSH, the MSH peptide analogues Nle⁴ -D-Phe⁷ -αMSH (NDP-MSH),ACTH₄₋₁₀ and ACTH₁₋₃₉. Following hormone treatment, the cells werewashed twice with phosphate buffered saline and intracellular cAMPextracted by lysing the cells with 1 mL of 60% ethanol. IntracellularcAMP concentrations were determined using an assay which measures theability of cAMP to displace (8-³ H) cAMP from a high affinity cAMPbinding protein (see Gilman, 1979, Proc. Natl. Acad. Sci. USA 67:305-312).

The results of these experiments are shown in FIGS. 11A through 11C. Theabscissa indicates the concentration of each hormone and the ordinateindicates the percentage of basal intracellular cAMP concentrationachieved by each treatment. Points indicate the mean of duplicateincubations; the standard error did not exceed 15% for any data point.FIG. 11A depicts the results of experiments using peptides found invivo; FIG. 11B depicts results found with γ-MSH variants; and FIG. 11Cshows results of synthetic melanocortin analogues. None of the peptidestested induced any change in intracellular cAMP in cells containing thevector alone. Cells expressing rat MC-3R responded strongly to everymelanotropic peptide containing the MSH code sequence His-Phe-Arg-Trp,with up to a 60-fold elevation of intracellular cAMP levels. EC₅₀ valuesranged from 1-50 nM. The most potent ligand and the one having thelowest EC₅₀ was found to be γMSH. The order of potency for the naturallyoccurring melanocortins was found to be:

    γ.sub.2 -MSH=γMSH>αMSH=ACTH.sub.1-39 >γ.sub.3 -MSH>des-acetyl-αMSH>ACTH.sub.4-10.

EC₅₀ values for these compounds are shown in Table II:

                  TABLE II                                                        ______________________________________                                        Agonist         EC.sub.50                                                     ______________________________________                                        NDP-MSH         1 × 10.sup.-9                                           γ.sub.1 -MSH                                                                            3 × 10.sup.-9                                           γ.sub.2 -MSH                                                                            3 × 10.sup.-9                                           α-MSH     4 × 10.sup.-9                                           ACTH.sub.1-39   4 × 10.sup.-9                                           γ.sub.3 -MSH                                                                            6 × 10.sup.-9                                           desacetyl-αMSH                                                                          8 × 10.sup.-9                                           ACTH.sub.4-10   1 × 10.sup.-7                                           ______________________________________                                    

Additionally, a synthetic melanocortin peptide (ORG2766), known to havethe greatest activity in vivo in stimulation of retention of learnedbehavior and in stimulation of neural regeneration, was unable tostimulate MC-3R-mediated cAMP production, and was also inactive as anantagonist. The results strongly indicate that this peptide does notbind to MC-3R protein.

The MC-4 receptor was cloned in a 1.9 kb HindIII genomic DNA fragmentafter PCR amplification of a lambda phage clone into pcDNAI/Neo(Invitrogen). This plasmid was stably introduced into human 293 cells bycalcium phosphate co-precipitation using standard techniques, andplasmid-containing cells selected in G418-containing media. Specificityof receptor-hormone binding was assayed using adenylate cylcase activityas described above. The MC-4 receptor was found to couple to adenylatecyclase activity having the following pattern of agonist affinity:

    NDP-MSH>des-acetyl-α-MSH>/=ACTH.sub.1-39 >/=α-MSH>>γ.sub.2 -MSH=ACTH.sub.4-10

whereas the synthetic ACTH₄₋₉ analogue ORG2766 showed no detectablebinding to the MC-4 receptor. The results of adenylate cyclase activityassays are shown in FIG. 12. EC₅₀ values for each of the tested MC-4receptor agonists are as shown in Table III:

                  TABLE III                                                       ______________________________________                                        Agonist        EC.sub.50                                                      ______________________________________                                        NDP-MSH        1.1 × 10.sup.-11 M                                       desacetyl-αMSH                                                                         4.9 × 10.sup.-10 M                                       ACTH.sub.1-39  6.8 × 10.sup.-10 M                                       α-MSH    1.5 × 10.sup.-9 M.sup.                                   γ.sub.2 -MSH                                                                           >10.sup.-7 M                                                   ACTH.sub.4-10  >10.sup.-7 M                                                   ______________________________________                                    

A 1.6 kb ApaI-HindIII fragment comprising the entire coding sequence ofthe mouse MC-5 melanocortin receptor disclosed in Example 2G above wascloned into the pcDNA/neo expression vector (Invitrogen) after PCRamplification of the lambda phage clone. This plasmid was stablyintroduced into human 293 cells by calcium phosphate co-precipitationusing standard techniques, and plasmid-containing cells selected in G418containing media. Specificity of receptor-hormone binding was assayedusing adenylate cylcase activity as described above. The MC-5 receptorwas found to couple to adenylate cyclase activity having the followingpattern of agonist affinity:

    α-MSH>βMSH>>γ-MSH

The results of adenylate cyclase activity assays (AC) and cAMP-dependentβ-galactosidase (β-gal) assay are shown in FIG. 13. EC₅₀ values for eachof the tested MC-5 receptor agonists are: α-MSH=1.7×10⁻⁹ M; andβMSH=5×10⁻⁹ M.

EXAMPLE 4 Melanocortin Analogue Binding to Mammalian MelanocortinReceptors

Recombinant cells prepared as described above in Example 3 were used tocharacterize receptor binding of two melanocortin analogues comprisingcyclic lactam heptapeptides.

The melanocortin receptor analogue SHU9119 has the following chemicalstructure: ##STR2##

The melanocortin receptor analogue MTII has the following chemicalstructure: ##STR3##

These analogues were prepared as described in Hruby et al. (1995, J.Med. Chem. 38: 3454-3461).

These analogues were tested for melanocortin receptor binding using acalorimetric assay system developed by some of the instant inventors(Chen et al., 1995, Analyt. Biochem. 226: 349-354) as follows. A seriesof concatamers of the synthetic oligonucleotide:

    5'-GAATTCGACGTCACAGTATGACGGCCATGG-3'                       (SEQ ID No:19)

was produced by self-annealing and ligation and a tandem tetramerobtained. This fragment was cloned upstream of a fragment of the humanvasoactive intestinal peptide (-93-+152; SEQ ID No.: 13; see Fink etal., 1988, Proc. Natl. Acad. Sci. USA 85: 6662-6666). This promoter wasthen cloned upstream of the β-galactosidase gene from E. coli. Theresulting plasmid construct is shown in FIG. 14.

Transient transfection of the pCRE/β-gal plasmid described above wasperformed as follows. Cells at between 40-60% confluency (correspondingto about 1.5 million cells/6 cm tissue culture plate) were incubatedwith Opti-MEM (Gibco BRL, Long Island, N.Y.) and then contacted with apCRE/β-gal-lipofectin complex which was prepared as follows. 3 μgplasmid DNA and 20 μL lipofectin reagent (Gibco) were each diluted into0.5 mL Opti-MEM media and then mixed together. This mixture wasincubated at room temperature for 15-20 min., and then the mixture (1mL) added to each 6 cm plate. Transfected plates were incubated at 37°C. for 5-24 h, after which the plates were washed and incubated withDMEM media (Gibco) and the cells split equally into a 96-well cultureplate.

To assay melanocortin receptor analogue binding, human 293 cellsexpressing each of the melanocortin receptors MC-1, MC-3, MC-4 and MC-5,and mouse Y1 cells expressing the MC-2 receptor, were transientlytransfected with pCRE/β-gal as described above and assayed as follows.Two days after transfection, cells were stimulated with hormonesspecific for each receptor or hormone analogue by incubation for 6 h at37° C. with a mixture comprising 10⁻¹² -10⁻⁶ M hormone or analogue, 0.1mg/mL bovine serum albumin and 0.1 mM isobutylmethylxanthine in DMEM.The effect of hormone or analogue binding was determined byβ-galactosidase assay according to the method of Felgner et al. (1994,J. Biol. Chem. 269: 2550-2561). Briefly, media was aspirated fromculture wells and 50 μL lysis buffer (0.25M Tris-HCl, pH 8/0.1%Triton-X100) added to each well. Cell lysis was enhanced by one round offreezing and thawing the cell/lysis buffer mixture. 10 μL aliquots weresampled from each well for protein determination using acommercially-available assay (BioRad, Hercules, Calif.). The remaining40 μL from each well was diluted with 40 μL phosphate bufferedsaline/0.5% BSA and 150 μL substrate buffer (60 mM sodium phosphate/1 mMMgCl₂ /10 mM KCl/5 mM β-mercaptoethanol/200 μg/mLo-nitrophenyl-β-D-galactopyranoside) added. Plates were incubated at 37°C. for 1 h and then absorbance at 405 nm determined using a 96-wellplate reader (Molecular Devices, Sunnyvale, Calif.). A series oftwo-fold dilutions from 20 ng of purified β-galactosidase protein (SigmaChemical Co, St. Louis, Mo.) were assayed in parallel in each experimentto enable conversion of OD₄₀₅ to known quantity of β-galactosidaseprotein.

The results of these experiments are shown in FIGS. 15A through 15D.This Figure shows the results of the β-galactosidase assay describedabove using cells expressing each of the MC-1, MC-3, MC-4 or MC-5receptors and contacted with αMSH or a variety of αMSH analogues,including SHIU9119. These results showed that SHU9119 had relativelyweak agonist activity for both the human MC-3 and MC-4 receptors.

These results demonstrated the development of a colorimetric assay forcAMP accumulation as the result of melanocortin receptor binding toagonists and antagonists.

The action of MTII, SHU9119, and the endogenous mouse agouti peptide asagonists or antagonists of rodent MC receptors was determined byexamining their ability to elevate intracellular cAMP in 293 cell linesexpressing the rat MC3-R or mouse MC4-R (expressed as IC₅₀ valuesrepresenting ligand concentrations required for half-maximal inhibitionof binding of ¹²⁵ I-(Nle⁴,D-Phe⁷) α-MSH tracer). Agonist/antagonistactivity was also shown by demonstrating inhibition of cAMP elevation bythe potent α-MSH analogue (Nle⁴, D-Phe⁷)α-MSH, using either acAMP-responsive β-galactosidase reporter construct as described above,or by direct adenyl cyclase assay as described in Example 3 (whereinEC₅₀ values represent ligand concentration required for half-maximalactivation of a cAMP-responsive β-galactosidase reporter). Competitionbinding experiments were determined as the amount of radioactivity boundin the presence of 5×10⁻⁶ M cold (Nle⁴, D-Phe⁷)α-MSH, and was typically3-5% of total counts bound.

In these experiments, murine agouti peptide was produced using abaculovirus system as described by Lu et al., (1994, Nature 371:799-802), with the modification that the agouti peptide was purifiedfrom baculovirus supernatants by 0.6M NaCl step clution from an EconoScation exchange column (BioRad). Agouti peptide used in these assays wasapproximately 60% pure.

Competition binding assays were performed to determine whether SHU9119had antagonist activity towards αMSH binding to either the MC-3 or MC-4receptors. These assays were performed as follows. Human 293 cells(100,000 cells/well in 24-well plates) expressing either the MC-3 orMC-4 receptors prepared as described above were incubated with asolution of 1 mg/mL BSA in PBS containing 100,000 cpm (3.1×10⁻¹⁰ M (¹²⁵I)(Nle⁴, D-Phe⁷)αMSH and varying concentrations of αMSH, (Nle⁴,D-Phe⁷)αMSH or SHU9119. Cells were incubated for 30 min at 37° C.,washed twice with PBS-BSA, lysed with 0.5 mL 0.5N NaOH, and countedusing a γ-counter to quantitate the amount of bound (¹²⁵ I)(Nle⁴,D-Phe⁷)αMSH. Control experiments showed non-specific binding to occur atabout 3-5% levels, and this was taken into account when analyzing theexperimental results.

The SHU9119 analogue was found to be a potent antagonist of both thehuman MC-3 and MC-4 receptors, as shown in FIGS. 16A through 16D. Theseassays showed pA₂ values of 8.3 and 9.3 for the human MC-3 and MC-4receptors, respectively, as determined using the method of Schild (1947,Brit. J. Pharmacol. 2: 189-206). In contrast, no significant alterationin IC₅₀ values was detected in binding experiments using this analoguewith either the MC-3 or MC-4 receptors (FIGS. 17A and 17B).

The activity of the MTII analogue was also assayed for melanocortinreceptor agonist activity. These results are shown in FIGS. 18A and 18B,and confirmed that the MTII analogue is a specific agonist of the MC-3and MC-4 receptors.

Specific competition of (Nle⁴, D-Phe⁷)α-MSH binding to the rat MC-3receptor by agouti peptide was observed, although accurate IC₅₀ valuescould not be determined because the peptide preparation was nothomogeneous (results not shown). Specific competition of αMSH activationof human MC-4R by agouti was previously disclosed (Lu et al., 1994,Nature 371: 799-802).

EXAMPLE 5 Feeding Behavior Effect of Melanocortin Analogue Binding inBrain

The results shown in Example 4 above suggested a role in the regulationof feeding behavior in mammalian brain for MC receptor agonists andantagonists, in view of the antagonist properties of the agouti peptideat the MC-3 and MC-4 receptors. The agouti peptide was known to causeobesity when expressed ectopically in the mouse, and has been found tobe a high affinity antagonist of the melanocyte stimulating hormonereceptor (MC-1R) and of the hypothalamic MC-4 receptor (see Lu et al.,ibid.). The former activity explained the inhibitory effect of theagouti peptide on eumelanin pigment synthesis. Similarly, it washypothesized by the inventors that agouti causes obesity in mice byantagonizing hypothalamic MC-4 receptors. The cyclic melanocortinanalogue, SHU9119, having been shown herein and elsewhere (Hruby et al.,ibid.) to be a specific, high affinity antagonist of the MC-3 and MC-4receptors, was tested for the effect of direct administration to mousebrain on feeding behavior in the animals. Intracerebroventricular (ICV)administration of SHU9119 was performed to avoid any complicationscaused by inhibition of peptide traverse of the blood-brain barrier.

Briefly, male C57B1/6J mice (18-29 g) were maintained on a normal 12hr/12 hr light dark cycle with food (Purina mouse chow) and water adlibitum. Animals were housed individually for 24 hrs, distributed intoexperimental and control groups, avoiding any bias as a function ofprior weight, then injected with vehicle or vehicle plus drug just priorto the onset of a 12 hr light or dark cycle. Fasted animals weredeprived of food from 18:00 to 10:30 hrs to stimulate feeding during thedaytime experimental period. Animals were lightly anesthetized withhalothane, and administered into one lateral ventricle 2 μL of asolution of artificial cerebrospinal fluid alone (acsf, comprising 130mM NaCl, 27mM NaHCO₃, 1.2 mM Na₂ HPO₄, 0.3 mM NaH₂ PO₄, 0.5 mM Na₂ SO₄,1.0 mM CaCl₂, 1.0 mM MgCl₂, and 2.5 mM KCl), or 6 nmol SHU9119 in acsf.Freehand injections were performed as described by Laursen and Belknap(1986, J. Pharmacol. Methods 16: 355-357) with some modifications. A 10μl luertip syringe (Hamilton 701 LT) was fitted with a 0.5 inch 27 gaugeneedle. Stiff Tygon tubing was slipped over the needle to expose 3 mM ofthe needle tip. The syringe was held at a 45° angle from the front ofthe skull with the bevel facing up. The coronal suture was found bylightly rubbing the needle over the skull. Maintaining the 45° angle,the needle was then inserted 1-2 mm lateral to the midline, using onlymild pressure to insert and remove the needle. The compounds indicatedin a 2 μl volume of acsf were administered slowly over approximately 15seconds, and the needle removed after 35 seconds. Animals were allowedto recover from anesthesia and placed into a cage containing apremeasured quantity of food pellets in a spill-free cup. Moribundanimals were not included in the study.

Stimulation of feeding by intracerebroventricular administration of themelanocortin antagonist SHU9119 is shown in FIGS. 19A through 19C.Curves show cumulative food intake as a function of time followingadministration of the substances shown. FIG. 19A shows stimulation offeeding by administration of SHU9119 just prior to lights off (19:00hrs) to C57B 1/6J mice fed ad libidum. FIG. 19B, in contrast, shows noeffect of morning (10:00 hrs) SHU9119 administration in C57B1/6J micefed ad libidum. FIG. 19C illustrates stimulation of daytime feeding bySHU9119 administration in fasted C57B1/6J mice. In deriving the datapoints comprising these Figures, food remaining was briefly removed andweighted indica time intervals indicated. Data points indicate the meanand bars indicate standard error. Significance of the effect over timewas determined by ANOVA with repeated measures. Significance of drugeffects at individual time points was determined by two-way ANOVA, andis indicated in each Figure (***=P<0.001, **=P<0.01, *=P<0.05).

These results demonstrated that ICV administration of SHU9119 into onelateral ventricle of the C57B 1/6J mouse just prior to lights out led toa mean 60% increase in food intake over 12 hrs (FIG. 19A; P<0.005). Incontrast, daytime food intake in animals fed ad libidum was notstimulated by administration of SHU9119 (FIG. 19B). SHU9119-treatmentdid, however, significantly stimulate daytime food intake in animalsfasted for 16 hrs prior to the experiment (FIG. 19C; P<0.001).Stimulation of feeding was evident at approximately two hrspost-treatment, and continued for 12 hrs, to produce a mean 34% in foodintake relative to vehicle-injected controls.

These results supported the hypothesis that agouti or SHU9119 stimulatefeeding by antagonizing MC receptors in the central nervous system. Tofurther test this hypothesis, a series of experiments were performedwherein MC receptor agonists were administered to animals primed byfasting to eat, to determine whether feeding in such animals could beinhibited by the MC receptor agonists. Animals were induced to feed byfood deprivation for 16 h prior to ICV administration of thenon-specific melanocortin agonist MTII. In these experiments, ICVinjections in male C57B1/6J mice (20-30 g) and the measurement of foodintake were as described above.

Results of these experiments are shown in FIGS. 20A through 20C. Incomparison to vehicle-injected animals, MTII was found to produce apotent inhibition of feeding within one hour after administration (FIG.20A) in a dose-responsive manner. Food intake was significantlyinhibited for up to four hours following administration (P<0.001) at thehighest dose administered (3 nmol), and decreased food intake continuedfor the next four hours with normal rates of food intake resuming atabout 8 hours after treatment. This dose-responsive inhibition offeeding had an IC₅₀ at the two hour time point of approximately 0.5 nmol(FIG. 20C). However, inhibition of feeding with 3 nmol MTII wascompletely blocked by co-administration of 6 nmol SHU9119 (FIG. 20B;P<0.001), demonstrating that the effect results specifically fromagonist binding to the MC-4 and/or MC-3 receptor.

Locomotor assays were performed to determine whether the effects onfeeding behavior observed in these mice were secondary to generalizedbehavioral effects caused by administration of these melanocortinanalogues. The effects of MTII on locomotor activity were tested byplacing vehicle or MTII-treated mice in sound and light-proof cagescontaining multiple light beam detectors. These assays were performed byfirst injecting 3 nmol MTII or acsf as described above. At three hours(2:45-3:25) post-injection, 12 mice were placed into 12 separate boxescontaining multiple infrared light sources and photodetectors. The boxeswere contained within separate ventilated light and sound attenuatingchambers (Coulboum model E10-20). Disruption of the infrared beams, witha 10 msec. resolution, was tallied independently for each one minutetime period in each cage. The results of these assays are shown in FIG.20D. Data points indicate the mean total activity (# of light breaks)for 6 animals in each experimental group. Four way ANOVA statisticalanalysis was used to analyze the data, and indicated an absence of asignificant difference among the two groups.

Inhibition of feeding by MTII could not be explained by any apparentbehavioral abnormalities, or any effect on arousal or locomotoractivity. MTII-treated animals appeared alert and exhibited no unusualbehavior relative to controls. At approximately three hours after ICVadministration, MTII-treated animals exhibited locomotor activity thatwas indistinguishable from vehicle-treated animals (FIG. 20D). Thehigher initial activity, indicative of exploratory behavior, andcontinued locomotion over a 15 min period was indistinguishable betweenthe two groups, indicating that the inhibition of feeding was not due todecreased locomotion or decreased arousal.

The administration of MTII also inhibited food intake in three othermodels of hyperphagia: the C57B1/6J-Lep^(ob) mouse, C57B1/6J-A^(Y)mouse, and NPY-injected C57B/1/6J mouse. FIG. 21A shows inhibition offeeding by intracerebroventricular administration of MTII in A^(Y) mice(females, 19-28 gms). FIG. 21B shows inhibition of feeding byintracerebroventricular administration of MTII in C57B1/6J mice(females, 21-25 gm) stimulated to feed by co-administration ofneuropeptide Y. FIG. 21C shows inhibition of feeding byintracerebroventricular administration of the MTII in ob/ob mice(females, 48-69 gms). FIG. 21D shows inhibition of feeding in ob/ob miceintraperitoneal administration of MTII (females, 40-45 gms). ICVinjections and measurement of food intake was performed as describedabove, with the exception of NPY treated animals, which were not fastedprior to experimentation. Animals treated intraperitoneally received 100μl of a 1 mM solution of MTII in saline, and vehicle injectionsconsisted of the same value of saline alone. Significance indicated forindividual time points, determined as described above, was for 3 nmolMTII vs. acsf (FIG. 21A), 1.18 nmol NPY vs. 1.18 nmol NPY+3 nmol MTII(FIG. 21B), 3 nmol MTII vs. acsf (FIG. 21C), and 100 nmol MTII vs.saline (FIG. 21D).

The hyperphagia in these models can be clearly seen by comparing the 12hr food intake following a fast in vehicle-injected C57B/1/6J (2.4 g,FIG. 19A), C57B1/6J-A^(Y) (3.7 g, FIG. 21A) and C57B1/6J-Lep^(ob) (3.7g. FIG. 21C) animals. As expected, MTII treatment inhibited food intakefollowing a 16 hr fast in the C57B1/6J-A^(Y) mouse (FIG. 21A; P<0.05).Interestingly, while food intake for the first four hours issignificantly inhibited relative to vehicle-injected animals, it is alsosignificantly less inhibited in the C57B1/6J-A^(Y) animal than in theC57B/1/6J animal given the same 3 nmol dose (compare, FIG. 20A versusFIG. 21A, 1-4 hrs; P<0.001). The decreased effectiveness of the agonistin the presence of the A^(Y) allele is consistent with the proposal thathis allele results in chronic expression of agouti peptide melanocortinantagonist in the brain.

MTII, upon co-administration, also significantly inhibited the profoundstimulation of feeding induced by neuropeptide Y (NPY), measured over athree hr period (FIG. 21C; P<0.005). Co-administration of anapproximately two-fold molar excess of MTII produced a 74% inhibition ofNPY-stimulated food intake at the three hour time point.

Finally, MTII also inhibited hyperphagia due to absence of leptin in theC57B1/6J-Lep^(ob) mouse (FIG. 21C; P<0.001). MTII blocked feeding aspotently (FIG. 20A), in contrast to the less potent inhibition describedabove for the C57B1/6J-A^(Y) mouse.

The C57B/1/6J-Lep^(ob) animal was also used to test the ability of MTIIto regulate feeding when administered peripherally. Moderate doses (100nmol) of MTII were inhibited feeding in the C57B1/6J-Lep^(ob) mouse(P<0.001) while low doses (10 nmol) did not (date not shown). Thekinetics were similar to those seen with ICV administration, with apotent inhibition of feeding for the first four hours. The 100-foldhigher dose required peripherally, as well as the similar kineticssuggest a primarily central nervous system mechanism of action of MTII.

These data show that melanocortinergic neurons exert a tonic inhibitionof feeding behavior, and that disruption of this signal leads tohyperphagia. With regard to the recently-discovered leptin hormone madeby adipocytes, which is generally expressed at elevated levels in obesehumans and rodents (such as the C57B1/6J-Lep^(ob) animal), theregulatory defect is understood to be an incapacity to respond properlyto the leptin hormone signal. The instant results indicate that themelanocortins act independently, or physiologically "downstream," fromthe leptin hormone/receptor interaction, because it has been shownherein that melanocortin receptor agonists can potently inhibit feedingin the C57B1/6J-Lep^(ob) animal.

These results suggest that MC receptor agonists and antagonists canaffect mammalian feeding behavior, and provide a means for determiningcandidate compounds for the development of effective pharmacologicalproducts directed towards alleviating such human ailments as obesity,anorexia and cachexia.

EXAMPLE 6 Use of MC Receptor-Expressing Recombinant Cells for ScreeningCompounds that Affect Feeding Behavior in Mammals

The results obtained in Example 6 indicated that cells expressing avariety of mammalian melanocortin receptors are useful forcharacterizing compounds as a first step towards developing MC receptoragonists and antagonists for controlling feeding behavior in mammals,particularly obesity and overweight disorders in general, as well asanorexia, cachexia and other failure-to-thrive disorders.

A panel of mammalian melanocortin receptor-expressing recombinant cellsare provided as described above in Example 3, wherein each member of thepanel comprises appropriate mammalian cells, such as human 293 cells,comprising a recombinant expression construct encoding the MC-1, MC-2(ACTH), MC-3, MC-4 or MC-5 receptor, the panel constructed to comprisecells functionally expressing each of these MC receptor proteins.

The panel is used as follows. Receptor agonist activity is assayed bytransient or stable expression of a protein which produces a metabolitereporter molecule in response to receptor binding by any of the MCreceptor proteins. An example of such a reporter system is therecombinant expression construct described in Example 4, wherein cAMPresponsive elements (CREs) are operatively linked to bacterially-derivedβ-galactosidase (β-gal) gene. In the event of receptor binding, cAMP isproduced in the mammalian cell, and the CRE induces β-gal expression.When co-incubated with a colorless substrate for β-gal, receptor bindingresults in conversion of the colorless substrate to a blue-coloredproduct, which can be easily scored visually or spectrophotometrically.Alternative reporter genes, such a luciferase, can also be used asreporter systems, provided that expression of the reportermolecule-producing protein is functionally linked to receptor binding ofa test compound. Alternatively, cAMP production resulting from MCreceptor binding can also be measured directly. Additionally, the cellpanel or membranes from these cells can be used for direct radioligandbinding assays.

Assay panels are arranged so that agonist activity can be identified,quantitated and correlated with expression of each MC receptor.Automated assays using such panels are also envisioned, whereby thequalitative and quantitative detection of a reporter metabolite isdetected in an array (such as a 96-well tissue culture plate) and thedata collected and assembled into a computer data-base or otheranalytical program.

Antagonist activity is detected by a modification of the above assay. Inthis assay, the inhibition of production of an amount of a knownreceptor agonist, specific for each receptor, is assayed in the presenceof a putative antagonist compound. Production of metabolite reportermolecules and their qualitative and quantitative detection is achievedas described above, and the specificity and potency of each antagonistcompound characterized with regard to the degree of inhibition achievedfor each receptor.

In view of the instant disclosure, MC-3/MC-4 receptor antagonists areexpected to be useful to inhibit food intake in a hungry animal, andMC-3/MC-4 receptor agonists are expected to be useful to increase foodintake in an animal. Alternative patterns of feeding behavior associatedwith different patterns of MC receptor agonist/antagonist activity canbe determined using this assay.

Compounds having agonist or antagonist activity with the MC-3 or MC-4receptors detected using this assay are further screened in vivo todetermine whether the observed receptor binding activity results inmodification of feeding behavior when administered to an animal. Inthese assays, the MC receptor binding compounds detected using the assayare administered intracerebroventricularly as described above in Example5 to animals after an overnight fast, to waking animals, or to animalsthat are not otherwise primed to be hungry. Feeding and locomotoractivity is monitored in these animals, and compounds affecting eatingbehavior (either by inhibiting feeding in otherwise hungry animals orstimulating feeding in otherwise sated animals) are selected for furtherdevelopment.

In addition, systemic administration of compounds found to be active byICV administration assays is used to screen such compounds for theability to cross the blood-brain barrier. Such compounds are also usefulas templates for modifications aimed at increasing the availability ofthese compounds in the brain after systemic administration, forincreasing bioactivity, or both.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 19                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..35                                                 #/function = "DegenerateRMATION:                                                             oligonucleot - #ide primer (sense)"                            #"The residue at positions 24 and 24 are                                                     inosine"                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #       35         SATY RCNNTKGACM GSTAC                                      - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..32                                                 #/function = "DegenerateRMATION:                                                             oligonucleot - #ide primer (antisense)"                        #"The residue at position 18 is inosine"                                      -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #          32      ANCC AGCAGASRYG AA                                         - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1260 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..14                                                 -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 15..959                                               -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 960..1260                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - TTCCTGACAA GACT ATG TCC ACT CAG GAG CCC CAG A - #AG AGT CTT CTG GGT           50                                                                                          Met S - #er Thr Gln Glu Pro Gln Lys Ser Leu Leu Gl - #y       #               10                                                            - TCT CTC AAC TCC AAT GCC ACC TCT CAC CTT GG - #A CTG GCC ACC AAC CAG           98                                                                          Ser Leu Asn Ser Asn Ala Thr Ser His Leu Gl - #y Leu Ala Thr Asn Gln           #         25                                                                  - TCA GAG CCT TGG TGC CTG TAT GTG TCC ATC CC - #A GAT GGC CTC TTC CTC          146                                                                          Ser Glu Pro Trp Cys Leu Tyr Val Ser Ile Pr - #o Asp Gly Leu Phe Leu           #     40                                                                      - AGC CTA GGG CTG GTG AGT CTG GTG GAG AAT GT - #G CTG GTT GTG ATA GCC          194                                                                          Ser Leu Gly Leu Val Ser Leu Val Glu Asn Va - #l Leu Val Val Ile Ala           # 60                                                                          - ATC ACC AAA AAC CGC AAC CTG CAC TCG CCC AT - #G TAT TAC TTC ATC TGC          242                                                                          Ile Thr Lys Asn Arg Asn Leu His Ser Pro Me - #t Tyr Tyr Phe Ile Cys           #                 75                                                          - TGC CTG GCC CTG TCT GAC CTG ATG GTA AGT GT - #C AGC ATC GTG CTG GAG          290                                                                          Cys Leu Ala Leu Ser Asp Leu Met Val Ser Va - #l Ser Ile Val Leu Glu           #             90                                                              - ACT ACT ATC ATC CTG CTG CTG GAG GTG GGC AT - #C CTG GTG GCC AGA GTG          338                                                                          Thr Thr Ile Ile Leu Leu Leu Glu Val Gly Il - #e Leu Val Ala Arg Val           #        105                                                                  - GCT TTG GTG CAG CAG CTG GAC AAC CTC ATT GA - #C GTG CTC ATC TGT GGC          386                                                                          Ala Leu Val Gln Gln Leu Asp Asn Leu Ile As - #p Val Leu Ile Cys Gly           #   120                                                                       - TCC ATG GTG TCC AGT CTC TGC TTC CTG GGC AT - #C ATT GCT ATA GAC CGC          434                                                                          Ser Met Val Ser Ser Leu Cys Phe Leu Gly Il - #e Ile Ala Ile Asp Arg           125                 1 - #30                 1 - #35                 1 -       #40                                                                           - TAC ATC TCC ATC TTC TAT GCG CTG CGT TAT CA - #C AGC ATC GTG ACG CTG          482                                                                          Tyr Ile Ser Ile Phe Tyr Ala Leu Arg Tyr Hi - #s Ser Ile Val Thr Leu           #               155                                                           - CCC AGA GCA CGA CGG GCT GTC GTG GGC ATC TG - #G ATG GTC AGC ATC GTC          530                                                                          Pro Arg Ala Arg Arg Ala Val Val Gly Ile Tr - #p Met Val Ser Ile Val           #           170                                                               - TCC AGC ACC CTC TTT ATC ACC TAC TAC AAG CA - #C ACA GCC GTT CTG CTC          578                                                                          Ser Ser Thr Leu Phe Ile Thr Tyr Tyr Lys Hi - #s Thr Ala Val Leu Leu           #       185                                                                   - TGC CTC GTC ACT TTC TTT CTA GCC ATG CTG GC - #A CTC ATG GCG ATT CTG          626                                                                          Cys Leu Val Thr Phe Phe Leu Ala Met Leu Al - #a Leu Met Ala Ile Leu           #   200                                                                       - TAT GCC CAC ATG TTC ACG AGA GCG TGC CAG CA - #C GTC CAG GGC ATT GCC          674                                                                          Tyr Ala His Met Phe Thr Arg Ala Cys Gln Hi - #s Val Gln Gly Ile Ala           205                 2 - #10                 2 - #15                 2 -       #20                                                                           - CAG CTC CAC AAA AGG CGG CGG TCC ATC CGC CA - #A GGC TTC TGC CTC AAG          722                                                                          Gln Leu His Lys Arg Arg Arg Ser Ile Arg Gl - #n Gly Phe Cys Leu Lys           #               235                                                           - GGT GCT GCC ACC CTT ACT ATC CTT CTG GGG AT - #T TTC TTC CTG TGC TGG          770                                                                          Gly Ala Ala Thr Leu Thr Ile Leu Leu Gly Il - #e Phe Phe Leu Cys Trp           #           250                                                               - GGC CCC TTC TTC CTG CAT CTC TTG CTC ATC GT - #C CTC TGC CCT CAG CAC          818                                                                          Gly Pro Phe Phe Leu His Leu Leu Leu Ile Va - #l Leu Cys Pro Gln His           #       265                                                                   - CCC ACC TGC AGC TGC ATC TTC AAG AAC TTC AA - #C CTC TTC CTC CTC CTC          866                                                                          Pro Thr Cys Ser Cys Ile Phe Lys Asn Phe As - #n Leu Phe Leu Leu Leu           #   280                                                                       - ATC GTC CTC AGC TCC ACT GTT GAC CCC CTC AT - #C TAT GCT TTC CGC AGC          914                                                                          Ile Val Leu Ser Ser Thr Val Asp Pro Leu Il - #e Tyr Ala Phe Arg Ser           285                 2 - #90                 2 - #95                 3 -       #00                                                                           - CAG GAG CTC CGC ATG ACA CTC AAG GAG GTG CT - #G CTG TGC TCC TGG              95 - #9                                                                      Gln Glu Leu Arg Met Thr Leu Lys Glu Val Le - #u Leu Cys Ser Trp               #               315                                                           - TGATCAGAGG GCGCTGGGCA GAGGGTGACA GTGATATCCA GTGGCCTGCA TC - #TGTGAGAC       1019                                                                          - CACAGGTACT CATCCCTTCC TGATCTCCAT TTGTCTAAGG GTCGACAGGA TG - #AGCTTTAA       1079                                                                          - AATAGAAACC CAGAGTGCCT GGGGCCAGGA GAAAGGGTAA CTGTGACTGC AG - #GGCTCACC       1139                                                                          - CAGGGCAGCT ACGGGAAGTG GAGGAGACAG GGATGGGAAC TCTAGCCCTG AG - #CAAGGGTC       1199                                                                          - AGACCACAGG CTCCTGAAGA GCTTCACCTC TCCCCACCTA CAGGCAACTC CT - #GCTCAAGC       1259                                                                          #             1260                                                            - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 315 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ser Thr Gln Glu Pro Gln Lys Ser Leu Le - #u Gly Ser Leu Asn Ser         #                 15                                                          - Asn Ala Thr Ser His Leu Gly Leu Ala Thr As - #n Gln Ser Glu Pro Trp         #             30                                                              - Cys Leu Tyr Val Ser Ile Pro Asp Gly Leu Ph - #e Leu Ser Leu Gly Leu         #         45                                                                  - Val Ser Leu Val Glu Asn Val Leu Val Val Il - #e Ala Ile Thr Lys Asn         #     60                                                                      - Arg Asn Leu His Ser Pro Met Tyr Tyr Phe Il - #e Cys Cys Leu Ala Leu         # 80                                                                          - Ser Asp Leu Met Val Ser Val Ser Ile Val Le - #u Glu Thr Thr Ile Ile         #                 95                                                          - Leu Leu Leu Glu Val Gly Ile Leu Val Ala Ar - #g Val Ala Leu Val Gln         #           110                                                               - Gln Leu Asp Asn Leu Ile Asp Val Leu Ile Cy - #s Gly Ser Met Val Ser         #       125                                                                   - Ser Leu Cys Phe Leu Gly Ile Ile Ala Ile As - #p Arg Tyr Ile Ser Ile         #   140                                                                       - Phe Tyr Ala Leu Arg Tyr His Ser Ile Val Th - #r Leu Pro Arg Ala Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Arg Ala Val Val Gly Ile Trp Met Val Ser Il - #e Val Ser Ser Thr Leu         #               175                                                           - Phe Ile Thr Tyr Tyr Lys His Thr Ala Val Le - #u Leu Cys Leu Val Thr         #           190                                                               - Phe Phe Leu Ala Met Leu Ala Leu Met Ala Il - #e Leu Tyr Ala His Met         #       205                                                                   - Phe Thr Arg Ala Cys Gln His Val Gln Gly Il - #e Ala Gln Leu His Lys         #   220                                                                       - Arg Arg Arg Ser Ile Arg Gln Gly Phe Cys Le - #u Lys Gly Ala Ala Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Thr Ile Leu Leu Gly Ile Phe Phe Leu Cy - #s Trp Gly Pro Phe Phe         #               255                                                           - Leu His Leu Leu Leu Ile Val Leu Cys Pro Gl - #n His Pro Thr Cys Ser         #           270                                                               - Cys Ile Phe Lys Asn Phe Asn Leu Phe Leu Le - #u Leu Ile Val Leu Ser         #       285                                                                   - Ser Thr Val Asp Pro Leu Ile Tyr Ala Phe Ar - #g Ser Gln Glu Leu Arg         #   300                                                                       - Met Thr Leu Lys Glu Val Leu Leu Cys Ser Tr - #p                             305                 3 - #10                 3 - #15                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1633 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..461                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 462..1415                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1416..1633                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - CCCGCATGTG GCCGCCCTCA ATGGAGGGCT CTGAGAACGA CTTTTAAAAC GC - #AGAGAAAA         60                                                                          - AGCTCCATTC TTCCCAGACC TCAGCGCAGC CCTGGCCCAG GAAGGGAGGA GA - #CAGAGGCC        120                                                                          - AGGACGGTCC AGAGGTGTCG AAATGTCCTG GGAACCTGAG CAGCAGCCAC CA - #GGGAAGAG        180                                                                          - GCAGGGAGGG AGCTGAGGAC CAGGCTTGGT TGTGAGAATC CCTGAGCCCA GG - #CGGTTGAT        240                                                                          - GCCAGGAGGT GTCTGGACTG GCTGGGCCAT GCCTGGGCTG ACCTGTCCAG CC - #AGGGAGAG        300                                                                          - GGTGTGAGGG CAGATCTGGG GGTGCCCAGA TGGAAGGAGG CAGGCATGGG GA - #CACCCAAG        360                                                                          - GCCCCCTGGC AGCACCATGA ACTAAGCAGG ACACCTGGAG GGGAAGAACT GT - #GGGGACCT        420                                                                          #GTG CAG        473TCCT TCCTGCTTCC TGGACAGGAC T ATG GCT                       #          Met Ala Val Gln                                                    #            1                                                                - GGA TCC CAG AGA AGA CTT CTG GGC TCC CTC AA - #C TCC ACC CCC ACA GCC          521                                                                          Gly Ser Gln Arg Arg Leu Leu Gly Ser Leu As - #n Ser Thr Pro Thr Ala           #  20                                                                         - ATC CCC CAG CTG GGG CTG GCT GCC AAC CAG AC - #A GGA GCC CGG TGC CTG          569                                                                          Ile Pro Gln Leu Gly Leu Ala Ala Asn Gln Th - #r Gly Ala Arg Cys Leu           #                 35                                                          - GAG GTG TCC ATC TCT GAC GGG CTC TTC CTC AG - #C CTG GGG CTG GTG AGC          617                                                                          Glu Val Ser Ile Ser Asp Gly Leu Phe Leu Se - #r Leu Gly Leu Val Ser           #             50                                                              - TTG GTG GAG AAC GCG CTG GTG GTG GCC ACC AT - #C GCC AAG AAC CGG AAC          665                                                                          Leu Val Glu Asn Ala Leu Val Val Ala Thr Il - #e Ala Lys Asn Arg Asn           #         65                                                                  - CTG CAC TCA CCC ATG TAC TGC TTC ATC TGC TG - #C CTG GCC TTG TCG GAC          713                                                                          Leu His Ser Pro Met Tyr Cys Phe Ile Cys Cy - #s Leu Ala Leu Ser Asp           #     80                                                                      - CTG CTG GTG AGC GGG ACG AAC GTG CTG GAG AC - #G GCC GTC ATC CTC CTG          761                                                                          Leu Leu Val Ser Gly Thr Asn Val Leu Glu Th - #r Ala Val Ile Leu Leu           #100                                                                          - CTG GAG GCC GGT GCA CTG GTG GCC CGG GCT GC - #G GTG CTG CAG CAG CTG          809                                                                          Leu Glu Ala Gly Ala Leu Val Ala Arg Ala Al - #a Val Leu Gln Gln Leu           #               115                                                           - GAC AAT GTC ATT GAC GTG ATC ACC TGC AGC TC - #C ATG CTG TCC AGC CTC          857                                                                          Asp Asn Val Ile Asp Val Ile Thr Cys Ser Se - #r Met Leu Ser Ser Leu           #           130                                                               - TGC TTC CTG GGC GCC ATC GCC GTG GAC CGC TA - #C ATC TCC ATC TTC TAC          905                                                                          Cys Phe Leu Gly Ala Ile Ala Val Asp Arg Ty - #r Ile Ser Ile Phe Tyr           #       145                                                                   - GCA CTG CGC TAC CAC AGC ATC GTG ACC CTG CC - #G CGG GCG CCG CGA GCC          953                                                                          Ala Leu Arg Tyr His Ser Ile Val Thr Leu Pr - #o Arg Ala Pro Arg Ala           #   160                                                                       - GTT GCG GCC ATC TGG GTG GCC AGT GTC GTC TT - #C AGC ACG CTC TTC ATC         1001                                                                          Val Ala Ala Ile Trp Val Ala Ser Val Val Ph - #e Ser Thr Leu Phe Ile           165                 1 - #70                 1 - #75                 1 -       #80                                                                           - GGC TAC TAC GAC CAC GTG GCC GTC CTG CTG TG - #C CTC GTG GTC TTC TTC         1049                                                                          Gly Tyr Tyr Asp His Val Ala Val Leu Leu Cy - #s Leu Val Val Phe Phe           #               195                                                           - CTG GCT ATG CTG GTG CTC ATG GCC GTG CTG GA - #C GTC CAC ATG CTG GCC         1097                                                                          Leu Ala Met Leu Val Leu Met Ala Val Leu As - #p Val His Met Leu Ala           #           210                                                               - CGG GCC TGC CAG CAC GCC CAG GGC ATC GCC CG - #G CTC CAC AAG AGG CAG         1145                                                                          Arg Ala Cys Gln His Ala Gln Gly Ile Ala Ar - #g Leu His Lys Arg Gln           #       225                                                                   - CGC CCG GTC CAC CAG GGC TTT GGC CTT AAA GG - #C GCT GTC ACC CTC ACC         1193                                                                          Arg Pro Val His Gln Gly Phe Gly Leu Lys Gl - #y Ala Val Thr Leu Thr           #   240                                                                       - ATC CTG CTG GGC ATT TTC TTC CTC TGC TGG GG - #C CCC TTC TTC CTG CAT         1241                                                                          Ile Leu Leu Gly Ile Phe Phe Leu Cys Trp Gl - #y Pro Phe Phe Leu His           245                 2 - #50                 2 - #55                 2 -       #60                                                                           - CTC ACA CTC ATC GTC CTC TGC CCC GAG CAC CC - #C ACG TGC GGC TGC ATC         1289                                                                          Leu Thr Leu Ile Val Leu Cys Pro Glu His Pr - #o Thr Cys Gly Cys Ile           #               275                                                           - TTC AAG AAC TTC AAC CTC TTT CTC GCC CTC AT - #C ATC TGC AAT GCC ATC         1337                                                                          Phe Lys Asn Phe Asn Leu Phe Leu Ala Leu Il - #e Ile Cys Asn Ala Ile           #           290                                                               - ATC GAC CCC CTC ATC TAC GCC TTC CAC AGC CA - #G GAG CTC CGC AGG ACG         1385                                                                          Ile Asp Pro Leu Ile Tyr Ala Phe His Ser Gl - #n Glu Leu Arg Arg Thr           #       305                                                                   - CTC AAG GAG GTG CTG ACA TGC TCC TGG TGA GC - #GCGGTGCA CGCGCTTTAA           1435                                                                          Leu Lys Glu Val Leu Thr Cys Ser Trp  *                                        #   315                                                                       - GTGTGCTGGG CAGAGGGAGG TGGTGATATT GTGGTCTGGT TCCTGTGTGA CC - #CTGGGCAG       1495                                                                          - TTCCTTACCT CCCTGGTCCC CGTTTGTCAA AGAGGATGGA CTAAATGATC TC - #TGAAAGTG       1555                                                                          - TTGAAGCGCG GACCCTTCTG GGCAGGGAGG GGTCCTGCAA AACTCCAGGC AG - #GACTTCTC       1615                                                                          #1633              AC                                                         - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  317 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Ala Val Gln Gly Ser Gln Arg Arg Leu Le - #u Gly Ser Leu Asn Ser         #                 15                                                          - Thr Pro Thr Ala Ile Pro Gln Leu Gly Leu Al - #a Ala Asn Gln Thr Gly         #             30                                                              - Ala Arg Cys Leu Glu Val Ser Ile Ser Asp Gl - #y Leu Phe Leu Ser Leu         #         45                                                                  - Gly Leu Val Ser Leu Val Glu Asn Ala Leu Va - #l Val Ala Thr Ile Ala         #     60                                                                      - Lys Asn Arg Asn Leu His Ser Pro Met Tyr Cy - #s Phe Ile Cys Cys Leu         # 80                                                                          - Ala Leu Ser Asp Leu Leu Val Ser Gly Thr As - #n Val Leu Glu Thr Ala         #                 95                                                          - Val Ile Leu Leu Leu Glu Ala Gly Ala Leu Va - #l Ala Arg Ala Ala Val         #           110                                                               - Leu Gln Gln Leu Asp Asn Val Ile Asp Val Il - #e Thr Cys Ser Ser Met         #       125                                                                   - Leu Ser Ser Leu Cys Phe Leu Gly Ala Ile Al - #a Val Asp Arg Tyr Ile         #   140                                                                       - Ser Ile Phe Tyr Ala Leu Arg Tyr His Ser Il - #e Val Thr Leu Pro Arg         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Pro Arg Ala Val Ala Ala Ile Trp Val Al - #a Ser Val Val Phe Ser         #               175                                                           - Thr Leu Phe Ile Gly Tyr Tyr Asp His Val Al - #a Val Leu Leu Cys Leu         #           190                                                               - Val Val Phe Phe Leu Ala Met Leu Val Leu Me - #t Ala Val Leu Asp Val         #       205                                                                   - His Met Leu Ala Arg Ala Cys Gln His Ala Gl - #n Gly Ile Ala Arg Leu         #   220                                                                       - His Lys Arg Gln Arg Pro Val His Gln Gly Ph - #e Gly Leu Lys Gly Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Val Thr Leu Thr Ile Leu Leu Gly Ile Phe Ph - #e Leu Cys Trp Gly Pro         #               255                                                           - Phe Phe Leu His Leu Thr Leu Ile Val Leu Cy - #s Pro Glu His Pro Thr         #           270                                                               - Cys Gly Cys Ile Phe Lys Asn Phe Asn Leu Ph - #e Leu Ala Leu Ile Ile         #       285                                                                   - Cys Asn Ala Ile Ile Asp Pro Leu Ile Tyr Al - #a Phe His Ser Gln Glu         #   300                                                                       - Leu Arg Arg Thr Leu Lys Glu Val Leu Thr Cy - #s Ser Trp                     305                 3 - #10                 3 - #15                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2012 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..693                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 694..1587                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1588..2012                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - ACAACACTTT ATATATATTT TTATAAATGT AAGGGGTACA AAGGTGCCAT TT - #TGTTACAT         60                                                                          - GGATATACCG TGTAGTGGTG AAGCCTGGGC TTTTAGTGTA TCTGTCATCA GA - #ATAACATA        120                                                                          - CGTGTTACCC ATAGGAATTT CTCATCACCC GCCCCCTCCA CCCTTCGAGT CT - #CCAATGTC        180                                                                          - CATTCCACAC TCTATATCCA CGTGTATGCA TATAGCTCCA CATATAAGTG AG - #AACATGTA        240                                                                          - GTATTTGACT TCCTCTTTCT GAGTTATTTC ACTTTGATAA TGGCCTCCAC TT - #CCATCCAT        300                                                                          - GTTGCTGCAA AAGACATGAC CTTATTCTTT TTGATAGCTG GGGAGTACTC CA - #TTGTGTAT        360                                                                          - ATGTACCACA TTTCTTTATC CATTCACCCA TTGAGAACAC TTAGTTGATT CC - #ATATCTTT        420                                                                          - GCTATTGTCA CTAGTGCTGC AATAAACATA CATGTGCAGG CTCCTTCTAA TA - #TACTGATT        480                                                                          - TATATTTTAT GGAGAGAGAT AGAGTTCTTA GCGAGTGTGC TGTTTATTTC TA - #GTGTACTT        540                                                                          - GCAACTAATA TTCTGTATAC TCCCTTTAGG TGATTGGAGA TTTAACTTAG AT - #CTCCAGCA        600                                                                          - AGTGCTACAA GAAGAAAAGA TCCTGAAGAA TCAATCAAGT TTCCGTGAAG TC - #AAGTCCAA        660                                                                          - GTAACATCCC CGCCTTAACC ACAAGCAGGA GAA ATG AAG CAC AT - #T ATC AAC TCG         714                                                                          # Met Lys His Ile Ile Asn Ser                                                 #5  1                                                                         - TAT GAA AAC ATC AAC AAC ACA GCA AGA AAT AA - #T TCC GAC TGT CCT CGT          762                                                                          Tyr Glu Asn Ile Asn Asn Thr Ala Arg Asn As - #n Ser Asp Cys Pro Arg           #         20                                                                  - TGT GTT TTG CCG GAG GAG ATA TTT TTC ACA AT - #T TCC ATT GTT GGA GTT          810                                                                          Cys Val Leu Pro Glu Glu Ile Phe Phe Thr Il - #e Ser Ile Val Gly Val           #     35                                                                      - TTG GAG AAT CTG ATC GTC CTG CTG GCT GTG TT - #C AAG AAT AAG AAT CTC          858                                                                          Leu Glu Asn Leu Ile Val Leu Leu Ala Val Ph - #e Lys Asn Lys Asn Leu           # 55                                                                          - CAG GCA CCC ATG TAC TTT TTC ATC TGT AGC TT - #G GCC ATA TCT GAT ATG          906                                                                          Gln Ala Pro Met Tyr Phe Phe Ile Cys Ser Le - #u Ala Ile Ser Asp Met           #                 70                                                          - CTG GGC AGC CTA TAT AAG ATC TTG GAA AAT AT - #C CTG ATC ATA TTG AGA          954                                                                          Leu Gly Ser Leu Tyr Lys Ile Leu Glu Asn Il - #e Leu Ile Ile Leu Arg           #             85                                                              - AAC ATG GGC ATA CTC AAG CCA CGT GGC AGT TT - #T GAA ACC ACA GCC CAT         1002                                                                          Asn Met Gly Ile Leu Lys Pro Arg Gly Ser Ph - #e Glu Thr Thr Ala His           #        100                                                                  - GAC ATC ATC GAC TCC CTG TTT CTG CTC TCC CG - #T CTT GGC TCC ATC TTC         1050                                                                          Asp Ile Ile Asp Ser Leu Phe Leu Leu Ser Ar - #g Leu Gly Ser Ile Phe           #   115                                                                       - GAC CTG CTC GTG ATT GCT GCG GAC CGC TAC AT - #C ACC ATC TTC CAC GCA         1098                                                                          Asp Leu Leu Val Ile Ala Ala Asp Arg Tyr Il - #e Thr Ile Phe His Ala           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - CTG CGG TAC CAC AGC ATC GTG ACC ATG CGC CG - #C ACT GTG GTG GTG CTT         1146                                                                          Leu Arg Tyr His Ser Ile Val Thr Met Arg Ar - #g Thr Val Val Val Leu           #               150                                                           - ACG GTC ATC TGG ACG TTC TGC ACG GGG ACT GG - #C ATC ACC ATG GTG ATC         1194                                                                          Thr Val Ile Trp Thr Phe Cys Thr Gly Thr Gl - #y Ile Thr Met Val Ile           #           165                                                               - TTC TCC CAT CAT GTG CCC CAC GTG ATC ACC TT - #C ACG TCG CTG TTC CCG         1242                                                                          Phe Ser His His Val Pro His Val Ile Thr Ph - #e Thr Ser Leu Phe Pro           #       180                                                                   - CTG ATG CTG GTC TTC ATC CTG TGC CTC TAT GT - #G CAC ATG TTC CTG CTG         1290                                                                          Leu Met Leu Val Phe Ile Leu Cys Leu Tyr Va - #l His Met Phe Leu Leu           #   195                                                                       - GCT CGA TGG CAC ACC AGG AAG ATC TCC ACC CT - #C CCC AGA GCC AAC ATG         1338                                                                          Ala Arg Trp His Thr Arg Lys Ile Ser Thr Le - #u Pro Arg Ala Asn Met           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - AAA GGG GCC ATG ACA CTG ACC ATC CTG CTC GG - #G GTC TTC ATC TTC TGC         1386                                                                          Lys Gly Ala Met Thr Leu Thr Ile Leu Leu Gl - #y Val Phe Ile Phe Cys           #               230                                                           - TGG GCC CCC TTT GTG CTT CAT GTC CTC TTG AT - #G ACA TTC TGC CCA AGT         1434                                                                          Trp Ala Pro Phe Val Leu His Val Leu Leu Me - #t Thr Phe Cys Pro Ser           #           245                                                               - AAC CCC TAC TGC GCC TGC TAC ATG TCT CTC TT - #C CAG GTG AAC GGC ATG         1482                                                                          Asn Pro Tyr Cys Ala Cys Tyr Met Ser Leu Ph - #e Gln Val Asn Gly Met           #       260                                                                   - TTG ATC ATG TGC AAT GCC GTC ATT GAC CCC TT - #C ATA TAT GCC TTC CGG         1530                                                                          Leu Ile Met Cys Asn Ala Val Ile Asp Pro Ph - #e Ile Tyr Ala Phe Arg           #   275                                                                       - AGC CCA GAG CTC AGG GAC GCA TTC AAA AAG AT - #G ATC TTC TGC AGC AGG         1578                                                                          Ser Pro Glu Leu Arg Asp Ala Phe Lys Lys Me - #t Ile Phe Cys Ser Arg           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - TAC TGG TAG AATGGCTGAT CCCTGGTTTT AGAATCCATG GGAATAACG - #T                 1627                                                                          Tyr Trp  *                                                                    - TGCCAAGTGC CAGAATAGTG TAACATTCCA ACAAATGCCA GTGCTCCTCA CT - #GGCCTTCC       1687                                                                          - TTCCCTAATG GATGCAAGGA TGACCCACCA GCTAGTGTTT CTGAATACTA TG - #GCCAGGAA       1747                                                                          - CAGTCTATTG TAGGGGCAAC TCTATTTGTG ACTGGACAGA TAAAACGTGT AG - #TAAAAGAA       1807                                                                          - GGATAGAATA CAAAGTATTA GGTACAAAAG TAATTAGGTT TGCATTACTT AT - #GACAAATG       1867                                                                          - CATTACTTTT GCACCAATCT AGTAAAACAG CAATAAAAAT TCAAGGGCTT TG - #GGCTAAGG       1927                                                                          - CAAAGACTTG CTTTCCTGTG GACATTAACA AGCCAGTTCT GAGGCGGCCT TT - #CCAGGTGG       1987                                                                          #             2012 TTTC AGAGT                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  297 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Lys His Ile Ile Asn Ser Tyr Glu Asn Il - #e Asn Asn Thr Ala Arg         #                 15                                                          - Asn Asn Ser Asp Cys Pro Arg Cys Val Leu Pr - #o Glu Glu Ile Phe Phe         #             30                                                              - Thr Ile Ser Ile Val Gly Val Leu Glu Asn Le - #u Ile Val Leu Leu Ala         #         45                                                                  - Val Phe Lys Asn Lys Asn Leu Gln Ala Pro Me - #t Tyr Phe Phe Ile Cys         #     60                                                                      - Ser Leu Ala Ile Ser Asp Met Leu Gly Ser Le - #u Tyr Lys Ile Leu Glu         # 80                                                                          - Asn Ile Leu Ile Ile Leu Arg Asn Met Gly Il - #e Leu Lys Pro Arg Gly         #                 95                                                          - Ser Phe Glu Thr Thr Ala His Asp Ile Ile As - #p Ser Leu Phe Leu Leu         #           110                                                               - Ser Arg Leu Gly Ser Ile Phe Asp Leu Leu Va - #l Ile Ala Ala Asp Arg         #       125                                                                   - Tyr Ile Thr Ile Phe His Ala Leu Arg Tyr Hi - #s Ser Ile Val Thr Met         #   140                                                                       - Arg Arg Thr Val Val Val Leu Thr Val Ile Tr - #p Thr Phe Cys Thr Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Thr Gly Ile Thr Met Val Ile Phe Ser His Hi - #s Val Pro His Val Ile         #               175                                                           - Thr Phe Thr Ser Leu Phe Pro Leu Met Leu Va - #l Phe Ile Leu Cys Leu         #           190                                                               - Tyr Val His Met Phe Leu Leu Ala Arg Trp Hi - #s Thr Arg Lys Ile Ser         #       205                                                                   - Thr Leu Pro Arg Ala Asn Met Lys Gly Ala Me - #t Thr Leu Thr Ile Leu         #   220                                                                       - Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Ph - #e Val Leu His Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Met Thr Phe Cys Pro Ser Asn Pro Tyr Cy - #s Ala Cys Tyr Met Ser         #               255                                                           - Leu Phe Gln Val Asn Gly Met Leu Ile Met Cy - #s Asn Ala Val Ile Asp         #           270                                                               - Pro Phe Ile Tyr Ala Phe Arg Ser Pro Glu Le - #u Arg Asp Ala Phe Lys         #       285                                                                   - Lys Met Ile Phe Cys Ser Arg Tyr Trp                                         #   295                                                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1108 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..132                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 133..1026                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1027..1106                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - GGGGCCAGAA AGTTCCTGCT TCAGAGCAGA AGATCTTCAG CAAGAACTAC AA - #AGAAGAAA         60                                                                          - AGATTCTGGA GAATCAATCA AGTTTCCTGT CAAGTTCCAG TAACGTTTCT GT - #CTTAACTG        120                                                                          - CACACAGGAA AG ATG AAA CAC ATT CTC AAT CTG TAT - # GAA AAC CTC AAC            168                                                                                        Met Lys Hi - #s Ile Leu Asn Leu Tyr Glu Asn Leu Asn             #             10                                                              - AGT ACA GCA AGA AAT AAC TCA GAC TGT CCT GC - #T GTG ATT TTG CCA GAA          216                                                                          Ser Thr Ala Arg Asn Asn Ser Asp Cys Pro Al - #a Val Ile Leu Pro Glu           #         25                                                                  - GAG ATA TTT TTC ACA GTA TCC ATT GTT GGG GT - #T TTG GAG AAC CTG ATG          264                                                                          Glu Ile Phe Phe Thr Val Ser Ile Val Gly Va - #l Leu Glu Asn Leu Met           #     40                                                                      - GTC CTT CTG GCT GTG GCC AAG AAT AAG ATG CT - #T CAG TCG CCC ATG TAC          312                                                                          Val Leu Leu Ala Val Ala Lys Asn Lys Met Le - #u Gln Ser Pro Met Tyr           # 60                                                                          - TTT TTC ATC TGC AGC TTG GCT ATT TCC GAT AT - #G CTG GGG AGC ATG TAC          360                                                                          Phe Phe Ile Cys Ser Leu Ala Ile Ser Asp Me - #t Leu Gly Ser Met Tyr           #                 75                                                          - AAG ATT TTG GAA AAC GTT CTG ATC ATG TTC AA - #A AAC ATG GGT TAC CTC          408                                                                          Lys Ile Leu Glu Asn Val Leu Ile Met Phe Ly - #s Asn Met Gly Tyr Leu           #             90                                                              - GAG CCT CGA GGC AGT TTT GAA AGC ACA GCA GA - #T GAT GTG GTG GAC TCC          456                                                                          Glu Pro Arg Gly Ser Phe Glu Ser Thr Ala As - #p Asp Val Val Asp Ser           #        105                                                                  - CTG TTC ATC CTC TCC CTT CTC GGC TCC ATC TG - #C AGC CTG TCT GTG ATT          504                                                                          Leu Phe Ile Leu Ser Leu Leu Gly Ser Ile Cy - #s Ser Leu Ser Val Ile           #   120                                                                       - GCC GCT GAC CGC TAC ACT ACA ATC TTC CAC GC - #T CTG CAG TAC CAC CGC          552                                                                          Ala Ala Asp Arg Tyr Thr Thr Ile Phe His Al - #a Leu Gln Tyr His Arg           125                 1 - #30                 1 - #35                 1 -       #40                                                                           - ATC ATG ACC CCC GCA CCG TGC CCT CGT CAT CT - #G ACG GTC CTC TGG CGA          600                                                                          Ile Met Thr Pro Ala Pro Cys Pro Arg His Le - #u Thr Val Leu Trp Arg           #               155                                                           - GGC TGC ACA GGC AGT GGC ATT ACC ATC GTG AC - #C TTC TCC CAT CAC GTC          648                                                                          Gly Cys Thr Gly Ser Gly Ile Thr Ile Val Th - #r Phe Ser His His Val           #           170                                                               - CCC ACA GTG ATC GCC TTC ACA GCG CTG TTC CC - #G CTG ATG CTG GCC TTC          696                                                                          Pro Thr Val Ile Ala Phe Thr Ala Leu Phe Pr - #o Leu Met Leu Ala Phe           #       185                                                                   - ATC CTG TGC CTC TAC GTG CAC ATG TTC CTG CT - #G GCC CGC TCC CAC ACC          744                                                                          Ile Leu Cys Leu Tyr Val His Met Phe Leu Le - #u Ala Arg Ser His Thr           #   200                                                                       - AGG AGG ACC CCC TCC CTT CCC AAA GCC AAC AT - #G AGA GGG GCC GTC ACA          792                                                                          Arg Arg Thr Pro Ser Leu Pro Lys Ala Asn Me - #t Arg Gly Ala Val Thr           205                 2 - #10                 2 - #15                 2 -       #20                                                                           - CTG ACT GTC CTG CTC GGG GTC TTC ATT TTC TG - #T TGG GCA CCC TTT GTC          840                                                                          Leu Thr Val Leu Leu Gly Val Phe Ile Phe Cy - #s Trp Ala Pro Phe Val           #               235                                                           - CTT CAT GTC CTC TTG ATG ACA TTC TGC CCA GC - #T GAC CCC TAC TGT GCC          888                                                                          Leu His Val Leu Leu Met Thr Phe Cys Pro Al - #a Asp Pro Tyr Cys Ala           #           250                                                               - TGC TAC ATG TCC CTC TTC CAG GTG AAT GGT GT - #G TTG ATC ATG TGT AAT          936                                                                          Cys Tyr Met Ser Leu Phe Gln Val Asn Gly Va - #l Leu Ile Met Cys Asn           #       265                                                                   - GCC ATC ATC GAC CCC TTC ATA TAT GCC TTT CG - #G AGC CCA GAG CTC AGG          984                                                                          Ala Ile Ile Asp Pro Phe Ile Tyr Ala Phe Ar - #g Ser Pro Glu Leu Arg           #   280                                                                       - GTC GCA TTC AAA AAG ATG GTT ATC TGC AAC TG - #T TAC CAG TAG                 #1026                                                                         Val Ala Phe Lys Lys Met Val Ile Cys Asn Cy - #s Tyr Gln  *                    285                 2 - #90                 2 - #95                           - AATGATTGGT CCCTGATTTT AGGAGCCACA GGGATATACT GTCAGGGACA GA - #GTAGCGTG       1086                                                                          #               1108GGA CT                                                    - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  297 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Lys His Ile Leu Asn Leu Tyr Glu Asn Le - #u Asn Ser Thr Ala Arg         #                 15                                                          - Asn Asn Ser Asp Cys Pro Ala Val Ile Leu Pr - #o Glu Glu Ile Phe Phe         #             30                                                              - Thr Val Ser Ile Val Gly Val Leu Glu Asn Le - #u Met Val Leu Leu Ala         #         45                                                                  - Val Ala Lys Asn Lys Met Leu Gln Ser Pro Me - #t Tyr Phe Phe Ile Cys         #     60                                                                      - Ser Leu Ala Ile Ser Asp Met Leu Gly Ser Me - #t Tyr Lys Ile Leu Glu         # 80                                                                          - Asn Val Leu Ile Met Phe Lys Asn Met Gly Ty - #r Leu Glu Pro Arg Gly         #                 95                                                          - Ser Phe Glu Ser Thr Ala Asp Asp Val Val As - #p Ser Leu Phe Ile Leu         #           110                                                               - Ser Leu Leu Gly Ser Ile Cys Ser Leu Ser Va - #l Ile Ala Ala Asp Arg         #       125                                                                   - Tyr Thr Thr Ile Phe His Ala Leu Gln Tyr Hi - #s Arg Ile Met Thr Pro         #   140                                                                       - Ala Pro Cys Pro Arg His Leu Thr Val Leu Tr - #p Arg Gly Cys Thr Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Gly Ile Thr Ile Val Thr Phe Ser His Hi - #s Val Pro Thr Val Ile         #               175                                                           - Ala Phe Thr Ala Leu Phe Pro Leu Met Leu Al - #a Phe Ile Leu Cys Leu         #           190                                                               - Tyr Val His Met Phe Leu Leu Ala Arg Ser Hi - #s Thr Arg Arg Thr Pro         #       205                                                                   - Ser Leu Pro Lys Ala Asn Met Arg Gly Ala Va - #l Thr Leu Thr Val Leu         #   220                                                                       - Leu Gly Val Phe Ile Phe Cys Trp Ala Pro Ph - #e Val Leu His Val Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Met Thr Phe Cys Pro Ala Asp Pro Tyr Cy - #s Ala Cys Tyr Met Ser         #               255                                                           - Leu Phe Gln Val Asn Gly Val Leu Ile Met Cy - #s Asn Ala Ile Ile Asp         #           270                                                               - Pro Phe Ile Tyr Ala Phe Arg Ser Pro Glu Le - #u Arg Val Ala Phe Lys         #       285                                                                   - Lys Met Val Ile Cys Asn Cys Tyr Gln                                         #   295                                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1338 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..297                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 298..1269                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1270..1338                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - GGCTGTAACT GTAGCAACCG GTGTTGGGTG GGGATGAGAA GAGACCAGAG AG - #AGAGAGGG         60                                                                          - TCAGAGCGAC AGGGGATGAG ACAGGCTGGT CAGAGTCTGC ACTGATTGTT GG - #AGACGCAA        120                                                                          - AGGAAAGTTT TTTCTATGTC TCCAACCTCC CCCTCCTCCC CCGTTTCTCT CT - #GGAGAAAC        180                                                                          - TAAAATGTAG ACTGGACAGC ATCCACAAGA GAAGCACCTA GAAGAAGATT TT - #TTTTTCCC        240                                                                          - AGCAGCTTGC TCAGGACCCT GCAGGAGCTG CAGCCGGAAC TGGTCCCGCC GA - #TAACC           297                                                                          - ATG AAC TCT TCC TGC TGC CCG TCC TCC TCT TA - #T CCG ACG CTG CCT AAC          345                                                                          Met Asn Ser Ser Cys Cys Pro Ser Ser Ser Ty - #r Pro Thr Leu Pro Asn           #                 15                                                          - CTC TCC CAG CAC CCT GCA GCC CCC TCT GCC AG - #C AAC CGG AGT GGC AGT          393                                                                          Leu Ser Gln His Pro Ala Ala Pro Ser Ala Se - #r Asn Arg Ser Gly Ser           #             30                                                              - GGG TTC TGC GAG CAG GTT TTC ATC AAG CCA GA - #G GTC TTC CTG GCA CTG          441                                                                          Gly Phe Cys Glu Gln Val Phe Ile Lys Pro Gl - #u Val Phe Leu Ala Leu           #         45                                                                  - GGC ATC GTC AGT CTG ATG GAA AAC ATC CTG GT - #G ATC CTG GCT GTG GTG          489                                                                          Gly Ile Val Ser Leu Met Glu Asn Ile Leu Va - #l Ile Leu Ala Val Val           #     60                                                                      - AGG AAC GGC AAC CTG CAC TCC CCC ATG TAC TT - #C TTC CTG CTG AGC CTG          537                                                                          Arg Asn Gly Asn Leu His Ser Pro Met Tyr Ph - #e Phe Leu Leu Ser Leu           # 80                                                                          - CTG CAG GCC GAC CTG CTG GTG AGC CTG TCC AA - #C TCC CTG GAG ACC ATC          585                                                                          Leu Gln Ala Asp Leu Leu Val Ser Leu Ser As - #n Ser Leu Glu Thr Ile           #                 95                                                          - ATG ATC GTG GTT ATC AAC AGC GAC TCC CTG AC - #C TTG GAG GAC CAA TTC          633                                                                          Met Ile Val Val Ile Asn Ser Asp Ser Leu Th - #r Leu Glu Asp Gln Phe           #           110                                                               - ATC CAG CAC ATG GAC AAC ATC TTC GAC TCT AT - #G ATC TGC ATC TCC CTG          681                                                                          Ile Gln His Met Asp Asn Ile Phe Asp Ser Me - #t Ile Cys Ile Ser Leu           #       125                                                                   - GTG GCC TCC ATC TGC AAC CTC CTG GCC ATC GC - #C GTG GAC AGG TAC GTC          729                                                                          Val Ala Ser Ile Cys Asn Leu Leu Ala Ile Al - #a Val Asp Arg Tyr Val           #   140                                                                       - ACC ATC TTC TAT GCC CTC CGT TAC CAC AGC AT - #C ATG ACG GTT AGG AAA          777                                                                          Thr Ile Phe Tyr Ala Leu Arg Tyr His Ser Il - #e Met Thr Val Arg Lys           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GCC CTC TCC TTG ATC GTG GCC ATC TGG GTC TG - #C TGT GGC ATC TGC GGC          825                                                                          Ala Leu Ser Leu Ile Val Ala Ile Trp Val Cy - #s Cys Gly Ile Cys Gly           #               175                                                           - GTG ATG TTC ATC GTC TAC TCC GAG AGC AAG AT - #G GTC ATC GTG TGC CTC          873                                                                          Val Met Phe Ile Val Tyr Ser Glu Ser Lys Me - #t Val Ile Val Cys Leu           #           190                                                               - ATC ACC ATG TTC TTC GCC ATG GTG CTC CTC AT - #G GGC ACC CTG TAC ATC          921                                                                          Ile Thr Met Phe Phe Ala Met Val Leu Leu Me - #t Gly Thr Leu Tyr Ile           #       205                                                                   - CAC ATG TTC CTC TTC GCC AGG CTG CAC GTC CA - #G CGC ATC GCG GCA CTG          969                                                                          His Met Phe Leu Phe Ala Arg Leu His Val Gl - #n Arg Ile Ala Ala Leu           #   220                                                                       - CCA CCT GCT GAC GGG CTA GCC CCG CAG CAG CA - #C TCG TGC ATG AAG GGG         1017                                                                          Pro Pro Ala Asp Gly Leu Ala Pro Gln Gln Hi - #s Ser Cys Met Lys Gly           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GCC GTC ACC ATC ACC ATC CTG CTG GGG GTT TT - #C ATC TTC TGC TGG GCG         1065                                                                          Ala Val Thr Ile Thr Ile Leu Leu Gly Val Ph - #e Ile Phe Cys Trp Ala           #               255                                                           - CCT TTC TTC CTC CAC CTG GTC CTC ATC ATC AC - #C TGC CCC ACC AAC CCC         1113                                                                          Pro Phe Phe Leu His Leu Val Leu Ile Ile Th - #r Cys Pro Thr Asn Pro           #           270                                                               - TAC TGC ATC TGC TAC ACG GCG CAC TTC AAC AC - #C TAC CTG GTT CTC ATC         1161                                                                          Tyr Cys Ile Cys Tyr Thr Ala His Phe Asn Th - #r Tyr Leu Val Leu Ile           #       285                                                                   - ATG TGC AAC TCT GTC ATC GAC CCC CTC ATC TA - #C GCC TTC CGC AGC CTG         1209                                                                          Met Cys Asn Ser Val Ile Asp Pro Leu Ile Ty - #r Ala Phe Arg Ser Leu           #   300                                                                       - GAG CTG CGA AAC ACC TTC AAG GAG ATT CTC TG - #C GGT TGC AAT GGC ATG         1257                                                                          Glu Leu Arg Asn Thr Phe Lys Glu Ile Leu Cy - #s Gly Cys Asn Gly Met           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAC GTG GGC TAG GAACCCCCGA GGAGGTGTTC CACGGCTAGC CA - #AGAGAGAA             1309                                                                          Asn Val Gly  *                                                                #          1338    AGAC ACAGAAGGG                                             - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH:  323 ami - #no acids                                              (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Met Asn Ser Ser Cys Cys Pro Ser Ser Ser Ty - #r Pro Thr Leu Pro Asn         #                 15                                                          - Leu Ser Gln His Pro Ala Ala Pro Ser Ala Se - #r Asn Arg Ser Gly Ser         #             30                                                              - Gly Phe Cys Glu Gln Val Phe Ile Lys Pro Gl - #u Val Phe Leu Ala Leu         #         45                                                                  - Gly Ile Val Ser Leu Met Glu Asn Ile Leu Va - #l Ile Leu Ala Val Val         #     60                                                                      - Arg Asn Gly Asn Leu His Ser Pro Met Tyr Ph - #e Phe Leu Leu Ser Leu         # 80                                                                          - Leu Gln Ala Asp Leu Leu Val Ser Leu Ser As - #n Ser Leu Glu Thr Ile         #                 95                                                          - Met Ile Val Val Ile Asn Ser Asp Ser Leu Th - #r Leu Glu Asp Gln Phe         #           110                                                               - Ile Gln His Met Asp Asn Ile Phe Asp Ser Me - #t Ile Cys Ile Ser Leu         #       125                                                                   - Val Ala Ser Ile Cys Asn Leu Leu Ala Ile Al - #a Val Asp Arg Tyr Val         #   140                                                                       - Thr Ile Phe Tyr Ala Leu Arg Tyr His Ser Il - #e Met Thr Val Arg Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Leu Ser Leu Ile Val Ala Ile Trp Val Cy - #s Cys Gly Ile Cys Gly         #               175                                                           - Val Met Phe Ile Val Tyr Ser Glu Ser Lys Me - #t Val Ile Val Cys Leu         #           190                                                               - Ile Thr Met Phe Phe Ala Met Val Leu Leu Me - #t Gly Thr Leu Tyr Ile         #       205                                                                   - His Met Phe Leu Phe Ala Arg Leu His Val Gl - #n Arg Ile Ala Ala Leu         #   220                                                                       - Pro Pro Ala Asp Gly Leu Ala Pro Gln Gln Hi - #s Ser Cys Met Lys Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ala Val Thr Ile Thr Ile Leu Leu Gly Val Ph - #e Ile Phe Cys Trp Ala         #               255                                                           - Pro Phe Phe Leu His Leu Val Leu Ile Ile Th - #r Cys Pro Thr Asn Pro         #           270                                                               - Tyr Cys Ile Cys Tyr Thr Ala His Phe Asn Th - #r Tyr Leu Val Leu Ile         #       285                                                                   - Met Cys Asn Ser Val Ile Asp Pro Leu Ile Ty - #r Ala Phe Arg Ser Leu         #   300                                                                       - Glu Leu Arg Asn Thr Phe Lys Glu Ile Leu Cy - #s Gly Cys Asn Gly Met         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Asn Val Gly                                                                 - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..30                                                 #/function = "DegenerateRMATION:                                                             oligonucleot - #ide primer (sense)"                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #           30     TAYT DYTTCATCTG                                            - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..30                                                 #/function = "DegenerateRMATION:                                                             oligonucleot - #ide primer (sense)"                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #           30     TAKA TGARGGGGTC                                            - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1671 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA to mRNA                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: 5'UTR                                                           (B) LOCATION: 1..393                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 394..1389                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: 3'UTR                                                           (B) LOCATION: 1390..1671                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - AGCTTCCGAG AGGCAGCCGA TGTGAGCATG TGCGCACAGA TTCGTCTCCC AA - #TGGCATGG         60                                                                          - CAGCTTCAAG GAAAATTATT TTGAACAGAC TTGAATGCAT AAGATTAAAG TT - #AAAGCAGA        120                                                                          - AGTGAGAACA AGAAAGCAAA GAGCAGACTC TTTCAACTGA GAATGAATAT TT - #TGAAGCCC        180                                                                          - AAGATTTTAA CGTGATGATG ATTAGAGTCG TACCTAAAAG AGACTAAAAA CT - #CCATGTCA        240                                                                          - AGCTCTGGAC TTGTGACATT TACTCACAGC AGGCATGGCA ATTTTAGCCT CA - #CAACTTTC        300                                                                          - AGACAGATAA AGACTTGGAG GAAATAACTG AGACGACTCC CTGACCCAGG AG - #GTTAAATC        360                                                                          - AATTCAGGGG GACACTGGAA TTCTCCTGCC AGC ATG GTG AAC TC - #C ACC CAC CGT         414                                                                          # Met Val Asn Ser Thr His Arg                                                 #5  1                                                                         - GGG ATG CAC ACT TCT CTG CAC CTC TGG AAC CG - #C AGC AGT TAC AGA CTG          462                                                                          Gly Met His Thr Ser Leu His Leu Trp Asn Ar - #g Ser Ser Tyr Arg Leu           #         20                                                                  - CAC AGC AAT GCC AGT GAG TCC CTT GGA AAA GG - #C TAC TCT GAT GGA GGG          510                                                                          His Ser Asn Ala Ser Glu Ser Leu Gly Lys Gl - #y Tyr Ser Asp Gly Gly           #     35                                                                      - TGC TAC GCG CAA CTT TTT GTC TCT CCT GAG GT - #G TTT GTG ACT CTG GGT          558                                                                          Cys Tyr Ala Gln Leu Phe Val Ser Pro Glu Va - #l Phe Val Thr Leu Gly           # 55                                                                          - GTG ATC AGC TTG TTG GAG AAT ATC TTA GAG AT - #T GTG GCA ATA GCC AAG          606                                                                          Val Ile Ser Leu Leu Glu Asn Ile Leu Glu Il - #e Val Ala Ile Ala Lys           #                 70                                                          - AAC AAG AAT CTG CAT TCA CCC ATG TAC TTT TT - #C ATC TGC AGC TTG GCT          654                                                                          Asn Lys Asn Leu His Ser Pro Met Tyr Phe Ph - #e Ile Cys Ser Leu Ala           #             85                                                              - GTG GCT GAT ATG CTG GTG AGC GTT TCA AAT GG - #A TCA GAA ACC ATT ATC          702                                                                          Val Ala Asp Met Leu Val Ser Val Ser Asn Gl - #y Ser Glu Thr Ile Ile           #        100                                                                  - ATC ACC CTA TTA AAC CGT ACA GAT ACG GAT GC - #A CAG AGT TTC ACA GTG          750                                                                          Ile Thr Leu Leu Asn Arg Thr Asp Thr Asp Al - #a Gln Ser Phe Thr Val           #   115                                                                       - AAT ATT GAT AAT GTC ATT GAC TCG GTG ATC TG - #T AGC TCC TTG CTT GCA          798                                                                          Asn Ile Asp Asn Val Ile Asp Ser Val Ile Cy - #s Ser Ser Leu Leu Ala           120                 1 - #25                 1 - #30                 1 -       #35                                                                           - TCC ATT TGC AGC CTG CTT TCA ATT GCA GTG GA - #C AGG TAC TTT ACT ATC          846                                                                          Ser Ile Cys Ser Leu Leu Ser Ile Ala Val As - #p Arg Tyr Phe Thr Ile           #               150                                                           - TTC TAT GCT CTC CAG TAC CAT AAC ATT ATG AC - #A GTT AAG CGG GTT GGG          894                                                                          Phe Tyr Ala Leu Gln Tyr His Asn Ile Met Th - #r Val Lys Arg Val Gly           #           165                                                               - ATC AGC ATA AGT TGT ATC TGG GCA GCT TGC AC - #G GTT TCA GGT ATT TTG          942                                                                          Ile Ser Ile Ser Cys Ile Trp Ala Ala Cys Th - #r Val Ser Gly Ile Leu           #       180                                                                   - TTC ATC ATT TAC TCA GAT AGT AGT GCT GTC AT - #C ATC TGC CTC ATC ACC          990                                                                          Phe Ile Ile Tyr Ser Asp Ser Ser Ala Val Il - #e Ile Cys Leu Ile Thr           #   195                                                                       - ATG TTC TTC ACC ATG CTG GCT CTC ATG GCT TC - #T CTC TAT GTC CAC CTG         1038                                                                          Met Phe Phe Thr Met Leu Ala Leu Met Ala Se - #r Leu Tyr Val His Leu           200                 2 - #05                 2 - #10                 2 -       #15                                                                           - TTC CTG ATG GCC AGG CTT CAC ATT AAG AGG AT - #T GCT GTC CTC CCC GGC         1086                                                                          Phe Leu Met Ala Arg Leu His Ile Lys Arg Il - #e Ala Val Leu Pro Gly           #               230                                                           - ACT GGT GCC ATC CGC CAA GGT GCC AAT ATG AA - #G GGA GCG ATT ACC TTG         1134                                                                          Thr Gly Ala Ile Arg Gln Gly Ala Asn Met Ly - #s Gly Ala Ile Thr Leu           #           245                                                               - ACC ATC CTG ATT GGC GTC TTT GTT GTC TGC TG - #G GCC CCA TTC TTC CTC         1182                                                                          Thr Ile Leu Ile Gly Val Phe Val Val Cys Tr - #p Ala Pro Phe Phe Leu           #       260                                                                   - CAC TTA ATA TTC TAC ATC TCT TGT CCT CAG AA - #T CCA TAT TGT GTG TGC         1230                                                                          His Leu Ile Phe Tyr Ile Ser Cys Pro Gln As - #n Pro Tyr Cys Val Cys           #   275                                                                       - TTC ATG TCT CAC TTT AAC TTG TAT CTC ATA CT - #G ATC ATG TGT AAT TCA         1278                                                                          Phe Met Ser His Phe Asn Leu Tyr Leu Ile Le - #u Ile Met Cys Asn Ser           280                 2 - #85                 2 - #90                 2 -       #95                                                                           - ATC ATC GAT CCT CTG ATT TAT GCA CTC CGG AG - #T CAA GAA CTG AGG AAA         1326                                                                          Ile Ile Asp Pro Leu Ile Tyr Ala Leu Arg Se - #r Gln Glu Leu Arg Lys           #               310                                                           - ACC TTC AAA GAG ATC ATC TCT TCC TAT CCC CT - #G GGA GGC CTT TGT GAC         1374                                                                          Thr Phe Lys Glu Ile Ile Ser Ser Tyr Pro Le - #u Gly Gly Leu Cys Asp           #           325                                                               - TTG TCT AGC AGA TAT TAAATGGGGA CAGAGCACGC AATATAGGA - #A CATCCATAAG         1429                                                                          Leu Ser Ser Arg Tyr                                                                   330                                                                   - AGACTTTTTC ACTCTTACCC TACCTGAATA TTCTACTTCT GCAACAGCTT TC - #TCTTCCGT       1489                                                                          - GTAGGGTACT GGTTGAGATA TCCATTGTGT AAATTTAAGC CTATGATTTT TA - #ATGAGAAA       1549                                                                          - AAATGCCCAG TCTCTGTATT ATTTCCAATC TCATGCTACT TTTTTGGCCA TA - #AAATATGA       1609                                                                          - ATCTATGTTA TAGGTTGTAG GCACTGTGGA TTTACAAAAA GAAAAGTCCT TA - #TTAAAAGA       1669                                                                          #            1671                                                             - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 332 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Met Val Asn Ser Thr His Arg Gly Met His Th - #r Ser Leu His Leu Trp         #                 15                                                          - Asn Arg Ser Ser Tyr Arg Leu His Ser Asn Al - #a Ser Glu Ser Leu Gly         #             30                                                              - Lys Gly Tyr Ser Asp Gly Gly Cys Tyr Ala Gl - #n Leu Phe Val Ser Pro         #         45                                                                  - Glu Val Phe Val Thr Leu Gly Val Ile Ser Le - #u Leu Glu Asn Ile Leu         #     60                                                                      - Glu Ile Val Ala Ile Ala Lys Asn Lys Asn Le - #u His Ser Pro Met Tyr         # 80                                                                          - Phe Phe Ile Cys Ser Leu Ala Val Ala Asp Me - #t Leu Val Ser Val Ser         #                 95                                                          - Asn Gly Ser Glu Thr Ile Ile Ile Thr Leu Le - #u Asn Arg Thr Asp Thr         #           110                                                               - Asp Ala Gln Ser Phe Thr Val Asn Ile Asp As - #n Val Ile Asp Ser Val         #       125                                                                   - Ile Cys Ser Ser Leu Leu Ala Ser Ile Cys Se - #r Leu Leu Ser Ile Ala         #   140                                                                       - Val Asp Arg Tyr Phe Thr Ile Phe Tyr Ala Le - #u Gln Tyr His Asn Ile         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met Thr Val Lys Arg Val Gly Ile Ser Ile Se - #r Cys Ile Trp Ala Ala         #               175                                                           - Cys Thr Val Ser Gly Ile Leu Phe Ile Ile Ty - #r Ser Asp Ser Ser Ala         #           190                                                               - Val Ile Ile Cys Leu Ile Thr Met Phe Phe Th - #r Met Leu Ala Leu Met         #       205                                                                   - Ala Ser Leu Tyr Val His Leu Phe Leu Met Al - #a Arg Leu His Ile Lys         #   220                                                                       - Arg Ile Ala Val Leu Pro Gly Thr Gly Ala Il - #e Arg Gln Gly Ala Asn         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Met Lys Gly Ala Ile Thr Leu Thr Ile Leu Il - #e Gly Val Phe Val Val         #               255                                                           - Cys Trp Ala Pro Phe Phe Leu His Leu Ile Ph - #e Tyr Ile Ser Cys Pro         #           270                                                               - Gln Asn Pro Tyr Cys Val Cys Phe Met Ser Hi - #s Phe Asn Leu Tyr Leu         #       285                                                                   - Ile Leu Ile Met Cys Asn Ser Ile Ile Asp Pr - #o Leu Ile Tyr Ala Leu         #   300                                                                       - Arg Ser Gln Glu Leu Arg Lys Thr Phe Lys Gl - #u Ile Ile Ser Ser Tyr         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Pro Leu Gly Gly Leu Cys Asp Leu Ser Ser Ar - #g Tyr                         #               330                                                           - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 978 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..975                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - ATG AAC TCC TCC TCC ACC CTG ACT GTA TTG AA - #T CTT ACC CTG AAC GCC           48                                                                          Met Asn Ser Ser Ser Thr Leu Thr Val Leu As - #n Leu Thr Leu Asn Ala           #                 15                                                          - TCA GAG GAT GGC ATT TTA GGA TCA AAT GTC AA - #G AAC AAG TCT TTG GCC           96                                                                          Ser Glu Asp Gly Ile Leu Gly Ser Asn Val Ly - #s Asn Lys Ser Leu Ala           #             30                                                              - TGT GAA GAA ATG GGC ATT GCC GTG GAG GTG TT - #C CTG ACC CTG GGT CTC          144                                                                          Cys Glu Glu Met Gly Ile Ala Val Glu Val Ph - #e Leu Thr Leu Gly Leu           #         45                                                                  - GTC AGC CTC TTA GAG AAC ATC CTG GTC ATT GG - #G GCC ATA GTA AAG AAC          192                                                                          Val Ser Leu Leu Glu Asn Ile Leu Val Ile Gl - #y Ala Ile Val Lys Asn           #     60                                                                      - AAA AAC CTG CAC TCA CCC ATG TAC TTC TTT GT - #G GGC AGC TTA GCC GTG          240                                                                          Lys Asn Leu His Ser Pro Met Tyr Phe Phe Va - #l Gly Ser Leu Ala Val           # 80                                                                          - GCC GAC ATG CTG GTG AGC ATG TCC AAT GCC TG - #G GAG ACT GTC ACC ATA          288                                                                          Ala Asp Met Leu Val Ser Met Ser Asn Ala Tr - #p Glu Thr Val Thr Ile           #                 95                                                          - TAC TTG CTA AAT AAT AAA CAC CTG GTG ATA GC - #C GAC ACC TTT GTG CGA          336                                                                          Tyr Leu Leu Asn Asn Lys His Leu Val Ile Al - #a Asp Thr Phe Val Arg           #           110                                                               - CAC ATC GAC AAC GTG TTC GAC TCC ATG ATC TG - #C ATC TCT GTG GTG GCC          384                                                                          His Ile Asp Asn Val Phe Asp Ser Met Ile Cy - #s Ile Ser Val Val Ala           #       125                                                                   - TCG ATG TGC AGT TTG CTG GCC ATT GCG GTG GA - #T AGG TAC ATC ACC ATC          432                                                                          Ser Met Cys Ser Leu Leu Ala Ile Ala Val As - #p Arg Tyr Ile Thr Ile           #   140                                                                       - TTC TAT GCC TTG CGC TAC CAC CAC ATC ATG AC - #C GCG AGG CGC TCG GGG          480                                                                          Phe Tyr Ala Leu Arg Tyr His His Ile Met Th - #r Ala Arg Arg Ser Gly           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GTG ATC ATC GCC TGC ATT TGG ACC TTC TGC AT - #A AGC TGC GGC ATT GTT          528                                                                          Val Ile Ile Ala Cys Ile Trp Thr Phe Cys Il - #e Ser Cys Gly Ile Val           #               175                                                           - TTC ATC ATC TAC TAT GAG TCC AAG TAT GTG AT - #C ATT TGC CTC ATC TCC          576                                                                          Phe Ile Ile Tyr Tyr Glu Ser Lys Tyr Val Il - #e Ile Cys Leu Ile Ser           #           190                                                               - ATG TTC TTC ACC ATG CTG TTC TTC ATG GTG TC - #T CTG TAT ATA CAC ATG          624                                                                          Met Phe Phe Thr Met Leu Phe Phe Met Val Se - #r Leu Tyr Ile His Met           #       205                                                                   - TTC CTC CTG GCC CGG AAC CAT GTC AAG CGG AT - #A GCA GCT TCC CCC AGA          672                                                                          Phe Leu Leu Ala Arg Asn His Val Lys Arg Il - #e Ala Ala Ser Pro Arg           #   220                                                                       - TAC AAC TCC GTG AGG CAA AGG ACC AGC ATG AA - #G GGG GCT ATT ACC CTC          720                                                                          Tyr Asn Ser Val Arg Gln Arg Thr Ser Met Ly - #s Gly Ala Ile Thr Leu           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - ACC ATG CTA CTG GGG ATT TTC ATT GTC TGC TG - #G TCT CCC TTC TTT CTT          768                                                                          Thr Met Leu Leu Gly Ile Phe Ile Val Cys Tr - #p Ser Pro Phe Phe Leu           #               255                                                           - CAC CTT ATC TTA ATG ATC TCC TGC CCT CAG AA - #C GTC TAC TGC TCT TGC          816                                                                          His Leu Ile Leu Met Ile Ser Cys Pro Gln As - #n Val Tyr Cys Ser Cys           #           270                                                               - TTT ATG TCT TAC TTC AAC ATG TAC CTT ATA CT - #C ATC ATG TGC AAC TCC          864                                                                          Phe Met Ser Tyr Phe Asn Met Tyr Leu Ile Le - #u Ile Met Cys Asn Ser           #       285                                                                   - GTG ATC GAT CCT CTC ATC TAC GCC CTC CGC AG - #C CAA GAG ATG CGG AGG          912                                                                          Val Ile Asp Pro Leu Ile Tyr Ala Leu Arg Se - #r Gln Glu Met Arg Arg           #   300                                                                       - ACC TTT AAG GAG ATC GTC TGT TGT CAC GGA TT - #C CGG CGA CCT TGT AGG          960                                                                          Thr Phe Lys Glu Ile Val Cys Cys His Gly Ph - #e Arg Arg Pro Cys Arg           305                 3 - #10                 3 - #15                 3 -       #20                                                                           # 978              AT TAA                                                     Leu Leu Gly Gly Tyr                                                                           325                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 325 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Met Asn Ser Ser Ser Thr Leu Thr Val Leu As - #n Leu Thr Leu Asn Ala         #                 15                                                          - Ser Glu Asp Gly Ile Leu Gly Ser Asn Val Ly - #s Asn Lys Ser Leu Ala         #             30                                                              - Cys Glu Glu Met Gly Ile Ala Val Glu Val Ph - #e Leu Thr Leu Gly Leu         #         45                                                                  - Val Ser Leu Leu Glu Asn Ile Leu Val Ile Gl - #y Ala Ile Val Lys Asn         #     60                                                                      - Lys Asn Leu His Ser Pro Met Tyr Phe Phe Va - #l Gly Ser Leu Ala Val         # 80                                                                          - Ala Asp Met Leu Val Ser Met Ser Asn Ala Tr - #p Glu Thr Val Thr Ile         #                 95                                                          - Tyr Leu Leu Asn Asn Lys His Leu Val Ile Al - #a Asp Thr Phe Val Arg         #           110                                                               - His Ile Asp Asn Val Phe Asp Ser Met Ile Cy - #s Ile Ser Val Val Ala         #       125                                                                   - Ser Met Cys Ser Leu Leu Ala Ile Ala Val As - #p Arg Tyr Ile Thr Ile         #   140                                                                       - Phe Tyr Ala Leu Arg Tyr His His Ile Met Th - #r Ala Arg Arg Ser Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Val Ile Ile Ala Cys Ile Trp Thr Phe Cys Il - #e Ser Cys Gly Ile Val         #               175                                                           - Phe Ile Ile Tyr Tyr Glu Ser Lys Tyr Val Il - #e Ile Cys Leu Ile Ser         #           190                                                               - Met Phe Phe Thr Met Leu Phe Phe Met Val Se - #r Leu Tyr Ile His Met         #       205                                                                   - Phe Leu Leu Ala Arg Asn His Val Lys Arg Il - #e Ala Ala Ser Pro Arg         #   220                                                                       - Tyr Asn Ser Val Arg Gln Arg Thr Ser Met Ly - #s Gly Ala Ile Thr Leu         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Met Leu Leu Gly Ile Phe Ile Val Cys Tr - #p Ser Pro Phe Phe Leu         #               255                                                           - His Leu Ile Leu Met Ile Ser Cys Pro Gln As - #n Val Tyr Cys Ser Cys         #           270                                                               - Phe Met Ser Tyr Phe Asn Met Tyr Leu Ile Le - #u Ile Met Cys Asn Ser         #       285                                                                   - Val Ile Asp Pro Leu Ile Tyr Ala Leu Arg Se - #r Gln Glu Met Arg Arg         #   300                                                                       - Thr Phe Lys Glu Ile Val Cys Cys His Gly Ph - #e Arg Arg Pro Cys Arg         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Leu Leu Gly Gly Tyr                                                                         325                                                           - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: misc.sub.-- - #feature                                          (B) LOCATION: 1..32                                                 #/function = "DegenerateRMATION:                                                             oligonucleot - #ide primer (antisense)"                        -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #           30     TATG ACGGCCATGG                                            __________________________________________________________________________

What we claim is:
 1. A method for characterizing a compound as a mammalian MC-3 or MC-4 melanocortin receptor agonist that inhibits feeding behavior in an animal, the method comprising the steps of:(a) providing a panel comprising a first mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the α-MSH receptor identified by Seq. ID Nos. 4 or 6, a second mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the ACTH receptor identified by Seq. ID Nos. 8 or 10, a third mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-3 receptor identified by Seq. ID No. 12, a fourth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-4 receptor identified by Seq. ID No. 16, and a fifth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-5 receptor identified by Seq ID No. 18, wherein each mammalian cell expresses the melanocotin receptor encoded by the recombinant expression construct comprising tie cell; (b) contacting each of the cells of the panel with a test compound to be characterized as an agonist of a mammalian melanocortin receptor; (c) detecting binding of the test compound to each of the mammalian melanocortin receptors by assaying for a metabolite produced in the cells that bind the compound, wherein detection of the metabolite indicates that the test compound is an agonist of the melanocortin receptor; (d) selecting the test compounds that are MC-3 or MC-4 melanocortin receptor agonists, and (e) determining whether the MC-3 or MC-4 melanocortin receptor agonist inhibits feeding behavior in an animal.
 2. The method of claim 1, wherein the metabolite detected in subpart (c) is cyclic AMP.
 3. The method of claim 1, each of the cells further comprising a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site operatively linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite.
 4. The method of claim 3, wherein the nucleic acid sequence encodes β-galactosidase.
 5. The method of claim 3, wherein the recombinant expression construct is pCRE/β-galactosidase.
 6. The method of claim 3, wherein the cells encoding each of the melanocortin receptors are cultured separately from each other and wherein the detectable metabolite produced by the protein encoded by the recombinant expression construct is produced by binding of the test compound to the mammalian melanocortin receptor encoded by each of the cells of the panel.
 7. The method of claim 1 wherein the test compound is an agonist of the MC-3 mammalian melanocortin receptor.
 8. The method of claim 1 wherein the test compound is an agonist of the MC-4 mammalian melanocortin receptor.
 9. The method of claim 3 wherein the test compound is an agonist of the MC-3 mammalian melanocortin receptor.
 10. The method of claim 3 wherein the test compound is an agonist of the MC-4 mammalian melanocortin receptor.
 11. A method for characterizing a compound as a mammalian MC-3 or MC-4 melanocortin receptor antagonist that stimulates feeding behavior in an animal, the method comprising the steps of:(a) providing a panel comprising a first mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the α-MSH receptor identified by Seq. ID Nos. 4 or 6, a second mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the ACTH receptor identified by Seq. ID Nos. 8 or 10, a third mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-3 receptor identified by Seq. ID No. 12, a fourth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-4 receptor identified by Seq. ID No. 16, and a fifth mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-5 receptor identified by Seq. ID No. 18, wherein each mammalian cell expresses the melanocortin receptor encoded by the recombinant expression construct comprising the cell, (b) contacting each of the cells of the panel with an agonist of the mammalian melanocortin receptor in an amount sufficient to produce a detectable amount of a metabolite produced in the cells that bind the agonist, in the presence or absence of a test compound to be characterized as an antagonist of a mammalian melanocortin receptor; (c) detecting the amount of the metabolite produced in each cell in the panel in the presence of the test compound with the amount of the metabolite produced in each cell in the panel in the absence, wherein the test compound is determined to be an antagonist of melanocortin binding if the amount of the metabolite produced in the presence of the test compound is less than the amount of the metabolite produced in the absence of the test compound; (d) selecting the test compounds that are MC-3 or MC-4 melanocortin receptor antagonist agonists, and (e) determining whether the MC-3 or MC-4 melanocortin receptor antagonist stimulates feeding behavior in an animal.
 12. The method of claim 11, wherein the metabolite detected in subpart (c) is cyclic AMP.
 13. The method of claim 11, each of the cells further comprising a recombinant expression construct encoding a cyclic AMP responsive element (CRE) transcription factor binding site operatively linked to a nucleic acid sequence encoding a protein capable of producing a detectable metabolite.
 14. The method of claim 13, wherein the nucleic acid sequence encodes β-galactosidase.
 15. The method of claim 13, wherein the recombinant expression construct is pCRE/β-galactosidase.
 16. The method of claim 13, wherein the cells encoding each of the melanocortin receptors are cultured separately from each other and wherein the detectable metabolite produced by the protein encoded by the recombinant expression construct is produced by binding of the test compound to the mammalian melanocortin receptor encoded by each of the cells of the panel.
 17. The method of claim 11 wherein the test compound is an antagonist of the MC-3 mammalian melanocortin receptor.
 18. The method of claim 11 wherein the test compound is an antagonist of the MC-4 mammalian melanocortin receptor.
 19. The method of claim 13 wherein the test compound is an antagonist of the MC-3 mammalian melanocortin receptor.
 20. The method of claim 13 wherein the test compound is an antagonist of the MC-4 mammalian melanocortin receptor.
 21. A biological screening panel for determining the mammalian melanocortin receptor agonist/antagonist profile of a test compound for the MC3 or MC-4 receptors, wherein the panel is used to identify compounds that modulate feeding behavior in an animal, the panel comprising a first mammalian cell consisting of a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-3 receptor identified by Seq. ID No. 12, and a second mammalian cell comprising a recombinant expression construct encoding a mammalian melanocortin receptor that is the MC-4 receptor identified by Seq. ID No. 16, wherein each mammalian cell expresses the melanocortin receptor encoded by the recombinant expression construct comprising the cell, and wherein compounds that modulate feeding behavior in a animal are selected from the MC-3/MC-4 mammalian melanocortin receptor agonists and antagonists. 